Eukaryotic chromatin is decorated with a wide range of reversible histone post-translational modifications (PTMs) that regulate all processes that require access to DNA, including transcription, DNA replication and DNA repair (Bannister and Kouzarides, 2011; Bowman and Poirier, 2015). Actively transcribed genes in all eukaryotes are enriched in monoubiquitinated histone H2B, which plays a non-degradative role in promoting transcription (Fleming et al., 2008; Weake and Workman, 2008) but whose mechanism of action remains poorly understood. Monoubiquitin is conjugated to H2B-K123 in yeast and H2B-K120 in humans, which lie near the C-terminus of histone H2B (Robzyk et al., 2000; Weake and Workman, 2008; West and Bonner, 1980). Monoubiquitination of histone H2B is highly dynamic (Henry et al., 2003), and the cycle of ubiquitination and subsequent deubiquitination is an important checkpoint for transcription elongation (Batta et al., 2011). H2B-K123 ubiquitination is required for methylation of histone H3K4 (Chandrasekharan et al., 2010; Dover et al., 2002) and H3K79 (Ng et al., 2002), two other marks correlated with actively transcribed genes. However, H2B-Ub also plays a role in promoting transcription that is independent of cross-talk with histone H3 methylation (Tanny et al., 2007). In addition to its role in transcription, H2B-Ub plays a role in replication fork progression, nucleosome assembly during DNA replication (Ng et al., 2002) and the DNA damage response (Giannattasio et al., 2005; Moyal et al., 2011; Uckelmann and Sixma, 2017). Dysregulation of histone H2B monoubiquitination has been linked to a variety of cancers (Cole et al., 2015; Espinosa, 2008; Hahn et al., 2012).

In the yeast, Saccharomyces cerevisiae, histone H2B-K123 is monoubiquitinated by the E2/E3 pair, Rad6/Bre1 (Hwang et al., 2003; Robzyk et al., 2000; Wood et al., 2003), and deubiquitinated by two deubiquitinating enzymes (DUBs): Ubp8 and Ubp10 (Daniel et al., 2004; Gardner et al., 2005; Henry et al., 2003). Both of these DUBs belong to the Ubiquitin Specific Protease (USP) class of cysteine proteases, which contain a characteristic USP catalytic domain (Komander et al., 2009). Ubp10 is a monomeric enzyme whereas Ubp8 is part of a four-protein subcomplex within the SAGA complex called the DUB module, which comprises Ubp8, Sgf11, Sus1, and Sgf73 (Henry et al., 2003; Köhler et al., 2010; Samara et al., 2010). Both yeast H2B-Ub DUBs are conserved in humans. USP36, the human homologue of Ubp10, can complement the effects of a ubp10 deletion on global H2B-Ub in yeast (Reed et al., 2015) and USP22, the homologue of Ubp8, is a subunit of human SAGA (Zhang et al., 2008). Yeast in which both Ubp10 and Ubp8 have been deleted showed a synergistic increase in the steady-state levels of global H2B-Ub, as well as growth defects (Emre et al., 2005). While the roles of Ubp10 and Ubp8 in regulating H2B deubiquitination are well-established, their respective contributions to chromatin-mediated processes are poorly understood.

Despite their shared substrate specificity, Ubp8 and Ubp10 appear to play distinct roles in vivo. Several studies have shown that SAGA/Ubp8 primarily acts on H2B-Ub near promoters and transcription start sites to promote transcription initiation by RNA polymerase II (Batta et al., 2011; Daniel et al., 2004; Schulze et al., 2011). Ubp10 was first identified for its role in regulating sub-telomeric gene silencing (Emre et al., 2005; Gardner et al., 2005; Kahana and Gottschling, 1999) and is recruited to silenced chromatin (Gardner et al., 2005). However, deletion of UBP10 alters expression of hundreds of yeast genes as well as H2B ubiquitination genome-wide (Gardner et al., 2005; Orlandi et al., 2004; Schulze et al., 2011), indicating that Ubp10 plays a global role beyond its function in subtelomeric transcriptional repression. Deletion of UBP8 also alters transcription of several hundred genes (Gardner et al., 2005), although an analysis of the data shows little correlation between the genes whose expression is impacted by ubp8 versus ubp10 deletion (Figure 1). The different impacts on transcription profiles suggest that these two H2B-Ub DUBs have distinct genomic targets. However, SAGA/Ubp8 was recently shown to be involved in transcription of all RNA polymerase II genes (Baptista et al., 2017; Warfield et al., 2017) and Ubp10 has been found in association with RNA polymerase II (Mao et al., 2014), suggesting that both DUBs may at least be present at all genes. A partial resolution of this conundrum comes from a genome-wide ChIP-on-chip study of H2B-Ub in ubp10 and ubp8 deletion strains (Schulze et al., 2011) which shows that loss of UBP8 results in an enrichment of H2B-Ub primarily near transcription start sites (TSS), whereas a ubp10 deletion strain shows broader enrichment of H2B-Ub in mid-coding regions of longer transcription units. The ChIP results suggest that Ubp8 and Ubp10 are required during transcription, but at different times and in different genic locations. However, it remains unclear how each of these factors produces these distinct profiles and what roles each enzyme plays during these processes.

Figure 1

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Ubiquitination of histone H2B has been reported to assist recruitment of the histone chaperone, FACT (Facilitates Chromatin Transcription) to active chromatin (Fleming et al., 2008). The yeast FACT complex is composed of a heterodimer of Spt16 and Pob3 that is assisted in vitro and in vivo by the DNA binding protein, Nhp6 (Brewster et al., 1998; Ruone et al., 2003; Schlesinger and Formosa, 2000; Wittmeyer and Formosa, 1995; Wittmeyer et al., 1999). FACT is reported to evict H2A/H2B heterodimers in front of the transcription machinery (Reinberg and Sims, 2006) and reassemble the heterodimers in the wake of RNA polymerase II to prevent cryptic transcription initiation (Fleming et al., 2008; Martin et al., 2018; Mason and Struhl, 2003; Pavri et al., 2006). The disruption of the H2B ubiquitination cycle or a mutation in the FACT subunit, Spt16, causes a defect in Pol II elongation (Fleming et al., 2008). In addition to roles in transcription, FACT and H2B-Ub are each also implicated in DNA replication (Formosa, 2012; Kurat et al., 2017; Trujillo and Osley, 2012). H2B-Ub at replication origins is thought to stabilize the parental nucleosomes after the passage of DNA polymerase (Trujillo and Osley, 2012). FACT and H2B-Ub play an important role in the progression of DNA replication, likely by maintaining chromatin stability and orchestrating nucleosome assembly on newly-synthesized DNA (Lin et al., 2014; Trujillo and Osley, 2012). It is clear that both FACT and H2B-Ub play a pivotal role in stabilizing and assembling nucleosomes in the wake of polymerases during replication and transcription. However, it is not known how FACT and H2B-Ub status affect one another to perform these functions.

We report here a novel role for the histone chaperone, FACT, in stimulating the H2B deubiquitination activity of Ubp10 on nucleosomes. We show that the rate of deubiquitination of yeast H2B-Ub is slower when incorporated into nucleosomes as compared to free H2A/H2B-Ub heterodimers, but that the addition of FACT reverses this block. This behavior is in marked contrast to the Ubp8/DUB module, which has robust activity on both heterodimers and intact nucleosomes and is not affected by FACT (Morgan et al., 2016). We show that a yeast strain with a FACT deficiency has elevated levels of H2B-Ub, indicating that FACT also stimulates deubiquitination of H2B in vivo. Deleting Ubp10, but not Ubp8, from a strain with mutated FACT conferred strong sensitivity to the DNA replication toxin, hydroxyurea (HU), and activated a cryptic transcription reporter. Our findings suggest that the differential effects of Ubp10 and Ubp8 on the distribution of H2B-Ub result from a global role for Ubp10 and FACT versus a local role of Ubp8/SAGA at promoters and transcription start sites. These observations have important implications for the way in which cycles of H2B ubiquitination and deubiquitination regulate nucleosome dynamics during transcription and DNA replication.

We have discovered a previously uncharacterized synergy between the H2B deubiquitinating enzyme, Ubp10, and the histone chaperone, FACT, that suggests a unifying model for the role of H2B ubiquitination and deubiquitination in nucleosome dynamics. The role of H2B monoubiquitination in recruiting FACT to nucleosomes and the importance of both FACT and H2B-Ub in transcription (Batta et al., 2011; Fleming et al., 2008; Weake and Workman, 2008) and in DNA replication (Lin et al., 2014; Trujillo and Osley, 2012), are well-established. However, the interplay between nucleosome dynamics, H2B deubiquitination and histone chaperones had not been explored. In this study, we report a role for the histone chaperone, FACT, in deubiquitinating H2B-Ub and show that Ubp10 depends on FACT to efficiently cleave H2B-Ub from nucleosomes (Figure 2). This behavior is unlike Ubp8/SAGA, which does not require FACT to deubiquitinate nucleosomes (Morgan et al., 2016). Consistent with our in vitro results, we find that a mutation that decreases the level of FACT (pob3-L78R) results in increased levels of H2B-Ub that are comparable to those caused by loss of Ubp10 (Figure 4). We also show that combining FACT mutations with loss of Ubp10, but not loss of Ubp8, causes sensitivity to hydroxyurea, suggesting a role for the FACT-Ubp10 collaboration in DNA replication. Similarly, combining FACT mutations with deletion of UBP10, but not UBP8, activates a cryptic promoter reporter gene, reflecting a role for Ubp10 in maintaining nucleosome occupancy during transcription. These combined in vivo and in vitro observations point to a role for Ubp10 and FACT in jointly maintaining chromatin organization and have important implications for the different cellular roles of the two H2B-Ub DUBs, Ubp8 and Ubp10.

Our results are consistent with a model in which Ubp10 plays a global role in regulating nucleosome dynamics in concert with FACT, while Ubp8 plays a more restricted role at sites of transcription initiation. This view is consistent with previous observations that Ubp8 deletion leads to higher levels of H2B-Ub in the vicinity of the +1 nucleosome, whereas Ubp10 deletions exhibit broader enrichment of H2B-Ub in gene bodies, particularly in longer open reading frames (Schulze et al., 2011). Ubp8 is targeted to genes in the context of the SAGA complex, a global transcription factor that associates with promoter regions of virtually all RNA polymerase II genes (Baptista et al., 2017; Baptista et al., 2018; Bonnet et al., 2014; Venters et al., 2011), which accounts for the observed pattern of H2B-Ub enrichment in ubp8 deletion strains (Schulze et al., 2011). The genome-wide pattern of H2B-Ub enrichment found in ubp10 deletion strains (Schulze et al., 2011) can be explained by a global function for Ubp10 in nucleosome assembly through its partnership with FACT. FACT has been implicated in transcription through its increased association with frequently transcribed genic regions (Feng et al., 2016; Mayer et al., 2010; Pathak et al., 2018; Saunders et al., 2003), but these same studies show that it is also abundant in intergenic regions. FACT physically interacts with both the MCM replicative helicase and with DNA polymerase α, and it promotes rapid deposition of nucleosomes in an in vitro replication system (Wittmeyer and Formosa, 1997; Yang et al., 2016; Yeeles et al., 2017). FACT is found in yeast cells at about two-thirds the abundance of nucleosomes and therefore is a global component of chromatin that participates in a broad range of chromatin-dependent processes (Gurova et al., 2018). These roles include restoring chromatin integrity after disruptions like transcription (Martin et al., 2018) but emerging evidence shows that FACT may have a less prominent role in proliferation of differentiated mammalian cells (Gurova et al., 2018; Shen et al., 2018). Collectively, these findings suggest, instead, that the primary role of FACT is to stabilize or maintain existing global chromatin architectures and promote transitions to new patterns during differentiation. The finding that Ubp10, but not Ubp8, plays an unanticipated role in DNA replication (Figures 4 and 5) and suppression of cryptic transcription (Figure 6) that overlaps with the role of FACT supports the idea that, like FACT, Ubp10 has a global function in maintaining stable chromatin architecture against a range of potential perturbations.

H2B ubiquitination has been proposed to stabilize chromatin as judged by a genome-wide reduction in nucleosome occupancy seen in mutants that lack Rad6, the E2 that ubiquitinates H2B, or in which wild type H2B is replaced by H2B-K123A, which cannot be ubiquitinated (Batta et al., 2011). Deletion of UBP8 enhances nucleosome occupancy genome-wide. However, the simple model that high global levels of H2B-Ub correlate with higher nucleosome occupancy is contradicted by the observation that highly transcribed genes have normal levels of nucleosome occupancy in a ubp8∆ mutant, even though these genes have markedly lower nucleosome occupancy in rad6∆ and H2B-K123A mutants (Batta et al., 2011). Our finding that a double ubp10∆/spt16-11 mutant activates a cryptic promoter points to a unique role for Ubp10 acting in concert with FACT to maintain nucleosome organization and is consistent with prior studies that suggest that cycles of both ubiquitination and deubiquitination are critical for nucleosome assembly during transcription (Batta et al., 2011; Fleming et al., 2008; Henry et al., 2003; Pavri et al., 2006).

In addition to its role in regulating global levels of H2B-Ub, Ubp10 regulates spreading of heterochromatic silencing at telomeres and at mating type loci (Emre et al., 2005; Gardner et al., 2005). This function is mediated by the N-terminus of Ubp10, which is recruited by the SIR complex to subtelomeric regions (Emre et al., 2005; Gardner et al., 2005; Zukowski et al., 2018). The silencing and global H2B ubiquitination functions of Ubp10 appear to be separable, as expression of N-terminal truncations of Ubp10 can restore wild type levels of H2B ubiquitination without restoring subtelomeric silencing (Reed et al., 2015). A recent study (Zukowski et al., 2018) reported that Sir2/Sir4, a subset of the SIR complex, stimulates the activity of GST-Ubp10 on both human nucleosomes and H2A/H2B-Ub heterodimers containing a hydrolyzable non-native linkage between ubiquitin and H2B. The observed stimulation was attributed to the ability of Sir2/Sir4 to recruit Ubp10 to chromatin, as has also been observed in vivo (Emre et al., 2005), as well as to a proposed allosteric stimulation of Ubp10 on both H2A/H2B-Ub heterodimers and on ubiquitinated nucleosomes (Zukowski et al., 2018). The effect observed in that study depended on residues 109–133 of Ubp10, which do not play a role in either Ubp10 catalytic activity on yeast H2A/H2B-Ub heterodimers, the ability to be stimulated by FACT (Figure 3), or FACT function in vivo (Figure 6—figure supplement 1). The effect of Sir2/Sir4 on Ubp10 thus appears to be restricted to its silencing-specific functions. Notably, human Usp15 also preferentially deubiquitinates H2A/H2B-Ub dimers and is stimulated by the splicing factor SART3 (Long et al., 2014), although in this case SART3 has the inverse effect: it enhances the activity of Usp15 on H2A/H2B-Ub but not on nucleosomes.

It is not clear why nucleosomes are such a poor substrate for Ubp10 as compared to H2A/H2B-Ub heterodimers. Ubp8 is targeted to nucleosomes in the context of the SAGA DUB module, in which Ubp8 forms a complex with Sgf11, Sus1 and Sgf73 (Henry et al., 2003; Köhler et al., 2010). The specificity of Ubp8 for H2B-K123 is conferred by Sgf11, which has a zinc finger domain with an arginine-rich patch (Köhler et al., 2010; Samara et al., 2010) that docks in the nucleosome acidic patch between histones H2A and H2B (Morgan et al., 2016). By contrast, Ubp10 is a monomeric enzyme that lacks accessory proteins that could promote binding to nucleosomes. However, Ubp10 has surprisingly high affinity for nucleosomes, binding with an apparent Kd of ~400 nM (Figure 3B). The wild-type Ubp8/DUB module, by contrast, does not bind detectably to nucleosomes by EMSA (Figure 3—figure supplement 3). The ability of Ubp10 to bind nucleosomes and the stimulation of Ubp10 enzymatic activity by FACT do not seem to be connected, as N-terminal deletions of Ubp10 that impair nucleosome binding are still stimulated by FACT (Figure 3B–D). Moreover, deletions within the N-terminal unstructured region of Ubp10 (Reed et al., 2015), which include residues important for recruitment to telomeres by the SIR complex (Kahana and Gottschling, 1999), have little or no effect on global H2B ubiquitination (Gardner et al., 2005). The significance of the ability of Ubp10 to bind nucleosomes, which was also observed by Zukowski et al. (2018), and the use of nucleosomes as substrates for deubiquitination therefore remains unresolved.

How does FACT stimulate the activity of Ubp10 on nucleosomes? While there is no direct evidence that FACT and Ubp10 work together in vivo, the synthetic genetic interaction and the biochemical cooperation that we observe is consistent with such a possibility. We propose that the structural changes that occur in canonical nucleosomes upon FACT binding are likely key to addressing this question, as the observed stimulation is specific to nucleosomal substrates. FACT can reorganize the nucleosome by disrupting histone/DNA contacts, leaving the nucleosome in an ‘open’ or ‘destabilized’ state (Chen et al., 2018; Kemble et al., 2015; McCullough et al., 2011) and by fully displacing H2A/H2B heterodimers (Belotserkovskaya et al., 2003; Chen et al., 2018; Hsieh et al., 2013; Orphanides et al., 1999; Wang et al., 2018; Xin et al., 2009). The most straightforward explanation for the effect of FACT on Ubp10 activity is therefore that FACT increases H2B deubiquitination by evicting H2A/H2B-Ub heterodimers, which are an excellent substrate for Ubp10 (Figures 2A and 3C). However, H2A-H2B displacement has been estimated to be limited to 20–50% of the total heterodimers (Orphanides et al., 1998; Xin et al., 2009) and requires the addition of Nhp6 to yeast Spt16-Pob3, whereas we observe complete deubiquitination, and no requirement for Nhp6. Dimer displacement can therefore only partially explain the effect of FACT. While it is not known whether ejected H2A/H2B heterodimers retain the H2B-K123 ubiquitin modification in vivo, our in vitro results clearly show that an H2A/H2B-Ub dimer bound to FACT can be rapidly deubiquitinated by Ubp10. Alternatively, FACT may stimulate Ubp10 by partly unwinding the DNA (Kemble et al., 2015; McCullough et al., 2011; Valieva et al., 2016), generating a hexasome, or yielding some other intermediate in nucleosome disassembly that either exposes surfaces that allow Ubp10 to interact more favorably with the H2B-Ub linkage or relieves steric clashes. In this second scenario, Ubp10 deubiquitinates H2B only after FACT has begun to destabilize the nucleosome, but without complete eviction of the dimer. In support of this idea, we find that the nucleosomes are intact following deubiquitination by Ubp10 (Figure 2—figure supplement 4). In either scenario, Ubp10 must deubiquitinate H2B-Ub at some point between the time the nucleosome is destabilized and before it is reassembled in the wake of either DNA or RNA polymerase. We think it is unlikely that FACT stimulates Ubp10 by recruiting it to the nucleosome, as we see no evidence that FACT enhances Ubp10 binding (Figure 3B and Figure 3—figure supplement 2). The enhancement of Ubp10 activity provides the first example of a physiologically relevant substrate that appears to be activated by FACT-mediated destabilization of nucleosomes. Further studies will be needed to unravel the molecular determinants governing Ubp10 substrate preference as well as the mechanism by which FACT activates deubiquitination of nucleosomes.

The coupling of Ubp10 and FACT activity provides a missing link between cycles of H2B ubiquitination and deubiquitination, and FACT activity and nucleosome disassembly and assembly that allows us to propose a global model for the role of H2B ubiquitination in chromatin dynamics (Figure 7). Ubiquitination of H2B by Rad6/Bre1 recruits FACT, which can either facilitate nucleosome disassembly or bind to nucleosomes that are already destabilized. Ubp10 then deubiquitinates H2B, enhancing the ability of FACT to promote reassembly of the nucleosome and/or reinsertion of a deubiquitinated H2A/H2B dimer. Deubiquitination of H2B could potentially favor nucleosome reassembly by enhancing release of FACT from its proposed checkpoint function (McCullough et al., 2018). The sequence of H2B ubiquitination, FACT-mediated nucleosome reorganization, then deubiquitination by Ubp10 and accelerated assembly could propel sequential assembly of nucleosomes, either in the wake of RNA polymerase or during DNA replication. This proposed sequence of events could also occur in humans, as well, given the conservation of all components of this sytem: RAD6B/RNF20/40 are the human E2/E3, USP36 is the homologue of Ubp10 and human FACT, Spt16/SSRP1, is the homologue of the yeast FACT complex, Spt16/Pob3/Nhp6. Our study provides a framework for understanding how H2B-Ub deubiquitination is coupled to the activity of the histone chaperone, FACT, in producing dynamic changes to nucleosome structure, and has exciting implications for understanding the mechanism by which dynamic cycles of ubiquitination and deubiquitination regulate chromatin organization.

Figure 7

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All data generated or analyzed during this study are included in the manuscript and supporting files.

1. 1. Bannister AJ
   2. Kouzarides T

   (2011) Regulation of chromatin by histone modifications

   Cell Research 21:381–395.

   https://doi.org/10.1038/cr.2011.22

   • PubMed
   • Google Scholar
2. 1. Baptista T
   2. Grünberg S
   3. Minoungou N
   4. Koster MJE
   5. Timmers HTM
   6. Hahn S
   7. Devys D
   8. Tora L

   (2017) SAGA is a general cofactor for RNA polymerase II transcription

   Molecular Cell 68:130–143.

   https://doi.org/10.1016/j.molcel.2017.08.016

   • PubMed
   • Google Scholar
3. 1. Baptista T
   2. Grünberg S
   3. Minoungou N
   4. Koster MJE
   5. Timmers HTM
   6. Hahn S
   7. Devys D
   8. Tora L

   (2018) SAGA is a general cofactor for RNA polymerase II transcription

   Molecular Cell 70:1163–1164.

   https://doi.org/10.1016/j.molcel.2018.06.007

   • PubMed
   • Google Scholar
4. 1. Batta K
   2. Zhang Z
   3. Yen K
   4. Goffman DB
   5. Pugh BF

   (2011) Genome-wide function of H2B ubiquitylation in promoter and genic regions

   Genes & Development 25:2254–2265.

   https://doi.org/10.1101/gad.177238.111

   • PubMed
   • Google Scholar
5. 1. Belotserkovskaya R
   2. Oh S
   3. Bondarenko VA
   4. Orphanides G
   5. Studitsky VM
   6. Reinberg D

   (2003) FACT facilitates transcription-dependent nucleosome alteration

   Science 301:1090–1093.

   https://doi.org/10.1126/science.1085703

   • PubMed
   • Google Scholar
6. 1. Bonnet J
   2. Wang CY
   3. Baptista T
   4. Vincent SD
   5. Hsiao WC
   6. Stierle M
   7. Kao CF
   8. Tora L
   9. Devys D

   (2014) The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription

   Genes & Development 28:1999–2012.

   https://doi.org/10.1101/gad.250225.114

   • PubMed
   • Google Scholar
7. 1. Bowman GD
   2. Poirier MG

   (2015) Post-translational modifications of histones that influence nucleosome dynamics

   Chemical Reviews 115:2274–2295.

   https://doi.org/10.1021/cr500350x

   • PubMed
   • Google Scholar
8. 1. Brewster NK
   2. Johnston GC
   3. Singer RA

   (1998) Characterization of the CP complex, an abundant dimer of Cdc68 and Pob3 proteins that regulates yeast transcriptional activation and chromatin repression

   Journal of Biological Chemistry 273:21972–21979.

   https://doi.org/10.1074/jbc.273.34.21972

   • PubMed
   • Google Scholar
9. 1. Chandrasekharan MB
   2. Huang F
   3. Chen YC
   4. Sun ZW

   (2010) Histone H2B C-terminal helix mediates trans-histone H3K4 methylation independent of H2B ubiquitination

   Molecular and Cellular Biology 30:3216–3232.

   https://doi.org/10.1128/MCB.01008-09

   • PubMed
   • Google Scholar
10. 1. Chen P
    2. Dong L
    3. Hu M
    4. Wang YZ
    5. Xiao X
    6. Zhao Z
    7. Yan J
    8. Wang PY
    9. Reinberg D
    10. Li M
    11. Li W
    12. Li G

    (2018) Functions of FACT in breaking the nucleosome and maintaining its integrity at the Single-Nucleosome level

    Molecular Cell 71:284–293.

    https://doi.org/10.1016/j.molcel.2018.06.020

    • PubMed
    • Google Scholar
11. 1. Cheung V
    2. Chua G
    3. Batada NN
    4. Landry CR
    5. Michnick SW
    6. Hughes TR
    7. Winston F

    (2008) Chromatin- and transcription-related factors repress transcription from within coding regions throughout the saccharomyces cerevisiae genome

    PLOS Biology 6:e277.

    https://doi.org/10.1371/journal.pbio.0060277

    • PubMed
    • Google Scholar
12. 1. Cole AJ
    2. Clifton-Bligh R
    3. Marsh DJ

    (2015) Histone H2B monoubiquitination: roles to play in human malignancy

    Endocrine-Related Cancer 22:T19–T33.

    https://doi.org/10.1530/ERC-14-0185

    • PubMed
    • Google Scholar
13. 1. Daniel JA
    2. Torok MS
    3. Sun ZW
    4. Schieltz D
    5. Allis CD
    6. Yates JR
    7. Grant PA

    (2004) Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription

    Journal of Biological Chemistry 279:1867–1871.

    https://doi.org/10.1074/jbc.C300494200

    • PubMed
    • Google Scholar
14. 1. Dover J
    2. Schneider J
    3. Tawiah-Boateng MA
    4. Wood A
    5. Dean K
    6. Johnston M
    7. Shilatifard A

    (2002) Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6

    Journal of Biological Chemistry 277:28368–28371.

    https://doi.org/10.1074/jbc.C200348200

    • PubMed
    • Google Scholar
15. 1. Dyer PN
    2. Edayathumangalam RS
    3. White CL
    4. Bao Y
    5. Chakravarthy S
    6. Muthurajan UM
    7. Luger K

    (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA

    Methods in Enzymology 375:23–44.

    https://doi.org/10.1016/S0076-6879(03)75002-2

    • PubMed
    • Google Scholar
16. 1. Elsässer SJ
    2. D'Arcy S

    (2012) Towards a mechanism for histone chaperones

    Biochimica Et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1819:211–221.

    https://doi.org/10.1016/j.bbagrm.2011.07.007

    • Google Scholar
17. 1. Emre NC
    2. Ingvarsdottir K
    3. Wyce A
    4. Wood A
    5. Krogan NJ
    6. Henry KW
    7. Li K
    8. Marmorstein R
    9. Greenblatt JF
    10. Shilatifard A
    11. Berger SL

    (2005) Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing

    Molecular Cell 17:585–594.

    https://doi.org/10.1016/j.molcel.2005.01.007

    • PubMed
    • Google Scholar
18. 1. Espinosa JM

    (2008) Histone H2B ubiquitination: the cancer connection

    Genes & Development 22:2743–2749.

    https://doi.org/10.1101/gad.1732108

    • PubMed
    • Google Scholar
19. 1. Evans TC
    2. Benner J
    3. Xu MQ

    (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element

    Protein Science 7:2256–2264.

    https://doi.org/10.1002/pro.5560071103

    • PubMed
    • Google Scholar
20. 1. Feng J
    2. Gan H
    3. Eaton ML
    4. Zhou H
    5. Li S
    6. Belsky JA
    7. MacAlpine DM
    8. Zhang Z
    9. Li Q

    (2016) Noncoding transcription is a driving force for nucleosome instability in spt16 mutant cells

    Molecular and Cellular Biology 36:1856–1867.

    https://doi.org/10.1128/MCB.00152-16

    • PubMed
    • Google Scholar
21. 1. Fleming AB
    2. Kao CF
    3. Hillyer C
    4. Pikaart M
    5. Osley MA

    (2008) H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation

    Molecular Cell 31:57–66.

    https://doi.org/10.1016/j.molcel.2008.04.025

    • PubMed
    • Google Scholar
22. 1. Formosa T

    (2012) The role of FACT in making and breaking nucleosomes

    Biochimica Et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1819:247–255.

    https://doi.org/10.1016/j.bbagrm.2011.07.009

    • Google Scholar
23. 1. Gardner RG
    2. Nelson ZW
    3. Gottschling DE

    (2005) Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin

    Molecular and Cellular Biology 25:6123–6139.

    https://doi.org/10.1128/MCB.25.14.6123-6139.2005

    • PubMed
    • Google Scholar
24. 1. Giannattasio M
    2. Lazzaro F
    3. Plevani P
    4. Muzi-Falconi M

    (2005) The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1

    Journal of Biological Chemistry 280:9879–9886.

    https://doi.org/10.1074/jbc.M414453200

    • PubMed
    • Google Scholar
25. 1. Gurova K
    2. Chang H-W
    3. Valieva ME
    4. Sandlesh P
    5. Studitsky VM

    (2018) Structure and function of the histone chaperone FACT – Resolving FACTual issues

    Biochimica Et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1861:892–904.

    https://doi.org/10.1016/j.bbagrm.2018.07.008

    • Google Scholar
26. 1. Hahn MA
    2. Dickson KA
    3. Jackson S
    4. Clarkson A
    5. Gill AJ
    6. Marsh DJ

    (2012) The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination

    Human Molecular Genetics 21:559–568.

    https://doi.org/10.1093/hmg/ddr490

    • PubMed
    • Google Scholar
27. 1. Henry KW
    2. Wyce A
    3. Lo WS
    4. Duggan LJ
    5. Emre NC
    6. Kao CF
    7. Pillus L
    8. Shilatifard A
    9. Osley MA
    10. Berger SL

    (2003) Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8

    Genes & Development 17:2648–2663.

    https://doi.org/10.1101/gad.1144003

    • PubMed
    • Google Scholar
28. 1. Hsieh FK
    2. Kulaeva OI
    3. Patel SS
    4. Dyer PN
    5. Luger K
    6. Reinberg D
    7. Studitsky VM

    (2013) Histone chaperone FACT action during transcription through chromatin by RNA polymerase II

    PNAS 110:7654–7659.

    https://doi.org/10.1073/pnas.1222198110

    • PubMed
    • Google Scholar
29. 1. Hwang WW
    2. Venkatasubrahmanyam S
    3. Ianculescu AG
    4. Tong A
    5. Boone C
    6. Madhani HD

    (2003) A conserved RING finger protein required for histone H2B monoubiquitination and cell size control

    Molecular Cell 11:261–266.

    https://doi.org/10.1016/S1097-2765(02)00826-2

    • PubMed
    • Google Scholar
30. 1. Jamai A
    2. Puglisi A
    3. Strubin M

    (2009) Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase

    Molecular Cell 35:377–383.

    https://doi.org/10.1016/j.molcel.2009.07.001

    • PubMed
    • Google Scholar
31. 1. Jasencakova Z
    2. Groth A

    (2010) Restoring chromatin after replication: how new and old histone marks come together

    Seminars in Cell & Developmental Biology 21:231–237.

    https://doi.org/10.1016/j.semcdb.2009.09.018

    • PubMed
    • Google Scholar
32. 1. Jbara M
    2. Maity SK
    3. Seenaiah M
    4. Brik A

    (2016) Palladium mediated rapid deprotection of N-Terminal cysteine under native chemical ligation conditions for the efficient preparation of synthetically challenging proteins

    Journal of the American Chemical Society 138:5069–5075.

    https://doi.org/10.1021/jacs.5b13580

    • PubMed
    • Google Scholar
33. 1. Jbara M
    2. Sun H
    3. Kamnesky G
    4. Brik A

    (2018) Chemical chromatin ubiquitylation

    Current Opinion in Chemical Biology 45:18–26.

    https://doi.org/10.1016/j.cbpa.2018.02.001

    • PubMed
    • Google Scholar
34. 1. Kahana A
    2. Gottschling DE

    (1999) DOT4 links silencing and cell growth in saccharomyces cerevisiae

    Molecular and Cellular Biology 19:6608–6620.

    https://doi.org/10.1128/MCB.19.10.6608

    • PubMed
    • Google Scholar
35. 1. Kaplan CD
    2. Laprade L
    3. Winston F

    (2003) Transcription elongation factors repress transcription initiation from cryptic sites

    Science 301:1096–1099.

    https://doi.org/10.1126/science.1087374

    • PubMed
    • Google Scholar
36. 1. Kemble DJ
    2. McCullough LL
    3. Whitby FG
    4. Formosa T
    5. Hill CP

    (2015) FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs

    Molecular Cell 60:294–306.

    https://doi.org/10.1016/j.molcel.2015.09.008

    • PubMed
    • Google Scholar
37. 1. Köhler A
    2. Zimmerman E
    3. Schneider M
    4. Hurt E
    5. Zheng N

    (2010) Structural basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase module

    Cell 141:606–617.

    https://doi.org/10.1016/j.cell.2010.04.026

    • PubMed
    • Google Scholar
38. 1. Komander D
    2. Clague MJ
    3. Urbé S

    (2009) Breaking the chains: structure and function of the deubiquitinases

    Nature Reviews Molecular Cell Biology 10:550–563.

    https://doi.org/10.1038/nrm2731

    • PubMed
    • Google Scholar
39. 1. Kurat CF
    2. Yeeles JTP
    3. Patel H
    4. Early A
    5. Diffley JFX

    (2017) Chromatin controls DNA replication origin selection, Lagging-Strand synthesis, and replication fork rates

    Molecular Cell 65:117–130.

    https://doi.org/10.1016/j.molcel.2016.11.016

    • PubMed
    • Google Scholar
40. 1. Lin CY
    2. Wu MY
    3. Gay S
    4. Marjavaara L
    5. Lai MS
    6. Hsiao WC
    7. Hung SH
    8. Tseng HY
    9. Wright DE
    10. Wang CY
    11. Hsu GS
    12. Devys D
    13. Chabes A
    14. Kao CF

    (2014) H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly

    PLOS Genetics 10:e1004667.

    https://doi.org/10.1371/journal.pgen.1004667

    • PubMed
    • Google Scholar
41. 1. Long L
    2. Thelen JP
    3. Furgason M
    4. Haj-Yahya M
    5. Brik A
    6. Cheng D
    7. Peng J
    8. Yao T

    (2014) The U4/U6 recycling factor SART3 has histone chaperone activity and associates with USP15 to regulate H2B deubiquitination

    Journal of Biological Chemistry 289:8916–8930.

    https://doi.org/10.1074/jbc.M114.551754

    • PubMed
    • Google Scholar
42. 1. Maity SK
    2. Jbara M
    3. Brik A

    (2016) Chemical and semisynthesis of modified histones

    Journal of Peptide Science 22:252–259.

    https://doi.org/10.1002/psc.2848

    • PubMed
    • Google Scholar
43. 1. Malone EA
    2. Clark CD
    3. Chiang A
    4. Winston F

    (1991) Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in saccharomyces cerevisiae

    Molecular and Cellular Biology 11:5710–5717.

    https://doi.org/10.1128/MCB.11.11.5710

    • PubMed
    • Google Scholar
44. 1. Mao P
    2. Meas R
    3. Dorgan KM
    4. Smerdon MJ

    (2014) UV damage-induced RNA polymerase II stalling stimulates H2B deubiquitylation

    PNAS 111:12811–12816.

    https://doi.org/10.1073/pnas.1403901111

    • PubMed
    • Google Scholar
45. 1. Martin BJE
    2. Chruscicki AT
    3. Howe LJ

    (2018) Transcription promotes the interaction of the FAcilitates chromatin transactions (FACT) Complex with nucleosomes in Saccharomyces cerevisiae

    Genetics 210:869–881.

    https://doi.org/10.1534/genetics.118.301349

    • PubMed
    • Google Scholar
46. 1. Mason PB
    2. Struhl K

    (2003) The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo

    Molecular and Cellular Biology 23:8323–8333.

    https://doi.org/10.1128/MCB.23.22.8323-8333.2003

    • PubMed
    • Google Scholar
47. 1. Mayer A
    2. Lidschreiber M
    3. Siebert M
    4. Leike K
    5. Söding J
    6. Cramer P

    (2010) Uniform transitions of the general RNA polymerase II transcription complex

    Nature Structural & Molecular Biology 17:1272–1278.

    https://doi.org/10.1038/nsmb.1903

    • PubMed
    • Google Scholar
48. 1. McCullough L
    2. Rawlins R
    3. Olsen A
    4. Xin H
    5. Stillman DJ
    6. Formosa T

    (2011) Insight into the mechanism of nucleosome reorganization from histone mutants that suppress defects in the FACT histone chaperone

    Genetics 188:835–846.

    https://doi.org/10.1534/genetics.111.128769

    • PubMed
    • Google Scholar
49. 1. McCullough LL
    2. Connell Z
    3. Xin H
    4. Studitsky VM
    5. Feofanov AV
    6. Valieva ME
    7. Formosa T

    (2018) Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization

    Journal of Biological Chemistry 293:6121–6133.

    https://doi.org/10.1074/jbc.RA117.000199

    • PubMed
    • Google Scholar
50. 1. Morgan MT
    2. Haj-Yahya M
    3. Ringel AE
    4. Bandi P
    5. Brik A
    6. Wolberger C

    (2016) Structural basis for histone H2B deubiquitination by the SAGA DUB module

    Science 351:725–728.

    https://doi.org/10.1126/science.aac5681

    • PubMed
    • Google Scholar
51. 1. Moyal L
    2. Lerenthal Y
    3. Gana-Weisz M
    4. Mass G
    5. So S
    6. Wang SY
    7. Eppink B
    8. Chung YM
    9. Shalev G
    10. Shema E
    11. Shkedy D
    12. Smorodinsky NI
    13. van Vliet N
    14. Kuster B
    15. Mann M
    16. Ciechanover A
    17. Dahm-Daphi J
    18. Kanaar R
    19. Hu MC
    20. Chen DJ
    21. Oren M
    22. Shiloh Y

    (2011) Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks

    Molecular Cell 41:529–542.

    https://doi.org/10.1016/j.molcel.2011.02.015

    • PubMed
    • Google Scholar
52. 1. Ng HH
    2. Xu RM
    3. Zhang Y
    4. Struhl K

    (2002) Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79

    The Journal of Biological Chemistry 277:34655–34657.

    https://doi.org/10.1074/jbc.C200433200

    • PubMed
    • Google Scholar
53. 1. Orlandi I
    2. Bettiga M
    3. Alberghina L
    4. Vai M

    (2004) Transcriptional profiling of ubp10 null mutant reveals altered subtelomeric gene expression and insurgence of oxidative stress response

    Journal of Biological Chemistry 279:6414–6425.

    https://doi.org/10.1074/jbc.M306464200

    • PubMed
    • Google Scholar
54. 1. Orphanides G
    2. LeRoy G
    3. Chang CH
    4. Luse DS
    5. Reinberg D

    (1998) FACT, a factor that facilitates transcript elongation through nucleosomes

    Cell 92:105–116.

    https://doi.org/10.1016/S0092-8674(00)80903-4

    • PubMed
    • Google Scholar
55. 1. Orphanides G
    2. Wu WH
    3. Lane WS
    4. Hampsey M
    5. Reinberg D

    (1999) The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins

    Nature 400:284–288.

    https://doi.org/10.1038/22350

    • PubMed
    • Google Scholar
56. 1. Pathak R
    2. Singh P
    3. Ananthakrishnan S
    4. Adamczyk S
    5. Schimmel O
    6. Govind CK

    (2018) Acetylation-Dependent recruitment of the FACT complex and its role in regulating pol II occupancy Genome-Wide in saccharomyces cerevisiae

    Genetics 209:743–756.

    https://doi.org/10.1534/genetics.118.300943

    • PubMed
    • Google Scholar
57. 1. Paull TT
    2. Johnson RC

    (1995) DNA looping by saccharomyces cerevisiae high mobility group proteins NHP6A/B. consequences for nucleoprotein complex assembly and chromatin condensation

    The Journal of Biological Chemistry 270:8744–8754.

    https://doi.org/10.1074/jbc.270.15.8744

    • PubMed
    • Google Scholar
58. 1. Pavri R
    2. Zhu B
    3. Li G
    4. Trojer P
    5. Mandal S
    6. Shilatifard A
    7. Reinberg D

    (2006) Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II

    Cell 125:703–717.

    https://doi.org/10.1016/j.cell.2006.04.029

    • PubMed
    • Google Scholar
59. 1. Ransom M
    2. Dennehey BK
    3. Tyler JK

    (2010) Chaperoning histones during DNA replication and repair

    Cell 140:183–195.

    https://doi.org/10.1016/j.cell.2010.01.004

    • PubMed
    • Google Scholar
60. 1. Reed BJ
    2. Locke MN
    3. Gardner RG

    (2015) A conserved deubiquitinating enzyme uses intrinsically disordered regions to scaffold multiple protein interaction sites

    Journal of Biological Chemistry 290:20601–20612.

    https://doi.org/10.1074/jbc.M115.650952

    • PubMed
    • Google Scholar
61. 1. Reinberg D
    2. Sims RJ

    (2006) De FACTo nucleosome dynamics

    Journal of Biological Chemistry 281:23297–23301.

    https://doi.org/10.1074/jbc.R600007200

    • PubMed
    • Google Scholar
62. 1. Robzyk K
    2. Recht J
    3. Osley MA

    (2000) Rad6-dependent ubiquitination of histone H2B in yeast

    Science 287:501–504.

    https://doi.org/10.1126/science.287.5452.501

    • PubMed
    • Google Scholar
63. 1. Ruone S
    2. Rhoades AR
    3. Formosa T

    (2003) Multiple Nhp6 molecules are required to recruit Spt16-Pob3 to form yFACT complexes and to reorganize nucleosomes

    Journal of Biological Chemistry 278:45288–45295.

    https://doi.org/10.1074/jbc.M307291200

    • PubMed
    • Google Scholar
64. 1. Samara NL
    2. Datta AB
    3. Berndsen CE
    4. Zhang X
    5. Yao T
    6. Cohen RE
    7. Wolberger C

    (2010) Structural insights into the assembly and function of the SAGA deubiquitinating module

    Science 328:1025–1029.

    https://doi.org/10.1126/science.1190049

    • PubMed
    • Google Scholar
65. 1. Saunders A
    2. Werner J
    3. Andrulis ED
    4. Nakayama T
    5. Hirose S
    6. Reinberg D
    7. Lis JT

    (2003) Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo

    Science 301:1094–1096.

    https://doi.org/10.1126/science.1085712

    • PubMed
    • Google Scholar
66. 1. Schlesinger MB
    2. Formosa T

    (2000)

    POB3 is required for both transcription and replication in the yeast saccharomyces cerevisiae

    Genetics 155:1593–1606.

    • PubMed
    • Google Scholar
67. 1. Schulze JM
    2. Hentrich T
    3. Nakanishi S
    4. Gupta A
    5. Emberly E
    6. Shilatifard A
    7. Kobor MS

    (2011) Splitting the task: Ubp8 and Ubp10 deubiquitinate different cellular pools of H2BK123

    Genes & Development 25:2242–2247.

    https://doi.org/10.1101/gad.177220.111

    • PubMed
    • Google Scholar
68. 1. Shen Z
    2. Formosa T
    3. Tantin D

    (2018) FACT inhibition blocks induction but not maintenance of pluripotency

    Stem Cells and Development 27:1693–1701.

    https://doi.org/10.1089/scd.2018.0150

    • PubMed
    • Google Scholar
69. 1. Simchen G
    2. Winston F
    3. Styles CA
    4. Fink GR

    (1984) Ty-mediated gene expression of the LYS2 and HIS4 genes of saccharomyces cerevisiae is controlled by the same SPT genes

    PNAS 81:2431–2434.

    https://doi.org/10.1073/pnas.81.8.2431

    • PubMed
    • Google Scholar
70. 1. Singh A
    2. Xu Y-J

    (2016) The cell killing mechanisms of hydroxyurea

    Genes 7:99.

    https://doi.org/10.3390/genes7110099

    • Google Scholar
71. 1. Southworth MW
    2. Amaya K
    3. Evans TC
    4. Xu MQ
    5. Perler FB

    (1999) Purification of proteins fused to either the amino or carboxy terminus of the mycobacterium xenopi gyrase A intein

    BioTechniques 27:110–120.

    https://doi.org/10.2144/99271st04

    • PubMed
    • Google Scholar
72. 1. Tanny JC
    2. Erdjument-Bromage H
    3. Tempst P
    4. Allis CD

    (2007) Ubiquitylation of histone H2B controls RNA polymerase II transcription elongation independently of histone H3 methylation

    Genes & Development 21:835–847.

    https://doi.org/10.1101/gad.1516207

    • PubMed
    • Google Scholar
73. 1. Trujillo KM
    2. Osley MA

    (2012) A role for H2B ubiquitylation in DNA replication

    Molecular Cell 48:734–746.

    https://doi.org/10.1016/j.molcel.2012.09.019

    • PubMed
    • Google Scholar
74. 1. Turco E
    2. Gallego LD
    3. Schneider M
    4. Köhler A

    (2015) Monoubiquitination of histone H2B is intrinsic to the Bre1 RING domain-Rad6 interaction and augmented by a second Rad6-binding site on Bre1

    Journal of Biological Chemistry 290:5298–5310.

    https://doi.org/10.1074/jbc.M114.626788

    • PubMed
    • Google Scholar
75. 1. Uckelmann M
    2. Sixma TK

    (2017) Histone ubiquitination in the DNA damage response

    DNA Repair 56:92–101.

    https://doi.org/10.1016/j.dnarep.2017.06.011

    • PubMed
    • Google Scholar
76. 1. Valieva ME
    2. Armeev GA
    3. Kudryashova KS
    4. Gerasimova NS
    5. Shaytan AK
    6. Kulaeva OI
    7. McCullough LL
    8. Formosa T
    9. Georgiev PG
    10. Kirpichnikov MP
    11. Studitsky VM
    12. Feofanov AV

    (2016) Large-scale ATP-independent nucleosome unfolding by a histone chaperone

    Nature Structural & Molecular Biology 23:1111–1116.

    https://doi.org/10.1038/nsmb.3321

    • PubMed
    • Google Scholar
77. 1. VanDemark AP
    2. Xin H
    3. McCullough L
    4. Rawlins R
    5. Bentley S
    6. Heroux A
    7. Stillman DJ
    8. Hill CP
    9. Formosa T

    (2008) Structural and functional analysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT subunits

    Journal of Biological Chemistry 283:5058–5068.

    https://doi.org/10.1074/jbc.M708682200

    • PubMed
    • Google Scholar
78. 1. Venters BJ
    2. Wachi S
    3. Mavrich TN
    4. Andersen BE
    5. Jena P
    6. Sinnamon AJ
    7. Jain P
    8. Rolleri NS
    9. Jiang C
    10. Hemeryck-Walsh C
    11. Pugh BF

    (2011) A comprehensive genomic binding map of gene and chromatin regulatory proteins in saccharomyces

    Molecular Cell 41:480–492.

    https://doi.org/10.1016/j.molcel.2011.01.015

    • PubMed
    • Google Scholar
79. 1. Wang T
    2. Liu Y
    3. Edwards G
    4. Krzizike D
    5. Scherman H
    6. Luger K

    (2018) The histone chaperone FACT modulates nucleosome structure by tethering its components

    Life Science Alliance 1:e201800107.

    https://doi.org/10.26508/lsa.201800107

    • PubMed
    • Google Scholar
80. 1. Warfield L
    2. Ramachandran S
    3. Baptista T
    4. Devys D
    5. Tora L
    6. Hahn S

    (2017) Transcription of nearly all yeast RNA polymerase II-Transcribed genes is dependent on transcription factor TFIID

    Molecular Cell 68:118–129.

    https://doi.org/10.1016/j.molcel.2017.08.014

    • PubMed
    • Google Scholar
81. 1. Weake VM
    2. Workman JL

    (2008) Histone ubiquitination: triggering gene activity

    Molecular Cell 29:653–663.

    https://doi.org/10.1016/j.molcel.2008.02.014

    • PubMed
    • Google Scholar
82. 1. West MH
    2. Bonner WM

    (1980) Histone 2B can be modified by the attachment of ubiquitin

    Nucleic Acids Research 8:4671–4680.

    https://doi.org/10.1093/nar/8.20.4671

    • PubMed
    • Google Scholar
83. 1. Winkler DD
    2. Muthurajan UM
    3. Hieb AR
    4. Luger K

    (2011) Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events

    Journal of Biological Chemistry 286:41883–41892.

    https://doi.org/10.1074/jbc.M111.301465

    • PubMed
    • Google Scholar
84. 1. Wittmeyer J
    2. Joss L
    3. Formosa T

    (1999) Spt16 and Pob3 of saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha

    Biochemistry 38:8961–8971.

    https://doi.org/10.1021/bi982851d

    • PubMed
    • Google Scholar
85. 1. Wittmeyer J
    2. Formosa T

    (1995) Identifying DNA replication complex components using protein affinity chromatography

    Methods in Enzymology 262:415–430.

    https://doi.org/10.1016/0076-6879(95)62033-8

    • PubMed
    • Google Scholar
86. 1. Wittmeyer J
    2. Formosa T

    (1997) The saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein

    Molecular and Cellular Biology 17:4178–4190.

    https://doi.org/10.1128/MCB.17.7.4178

    • PubMed
    • Google Scholar
87. 1. Wood A
    2. Krogan NJ
    3. Dover J
    4. Schneider J
    5. Heidt J
    6. Boateng MA
    7. Dean K
    8. Golshani A
    9. Zhang Y
    10. Greenblatt JF
    11. Johnston M
    12. Shilatifard A

    (2003) Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter

    Molecular Cell 11:267–274.

    https://doi.org/10.1016/S1097-2765(02)00802-X

    • PubMed
    • Google Scholar
88. 1. Xin H
    2. Takahata S
    3. Blanksma M
    4. McCullough L
    5. Stillman DJ
    6. Formosa T

    (2009) yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement

    Molecular Cell 35:365–376.

    https://doi.org/10.1016/j.molcel.2009.06.024

    • PubMed
    • Google Scholar
89. 1. Yang J
    2. Zhang X
    3. Feng J
    4. Leng H
    5. Li S
    6. Xiao J
    7. Liu S
    8. Xu Z
    9. Xu J
    10. Li D
    11. Wang Z
    12. Wang J
    13. Li Q

    (2016) The histone chaperone FACT contributes to DNA Replication-Coupled nucleosome assembly

    Cell Reports 16:3414.

    https://doi.org/10.1016/j.celrep.2016.08.070

    • PubMed
    • Google Scholar
90. 1. Yang S
    2. Liu L
    3. Cao C
    4. Song N
    5. Wang Y
    6. Ma S
    7. Zhang Q
    8. Yu N
    9. Ding X
    10. Yang F
    11. Tian S
    12. Zhang K
    13. Sun T
    14. Yang J
    15. Yao Z
    16. Wu S
    17. Shi L

    (2018) USP52 acts as a deubiquitinase and promotes histone chaperone ASF1A stabilization

    Nature Communications 9:1285.

    https://doi.org/10.1038/s41467-018-03588-z

    • PubMed
    • Google Scholar
91. 1. Yeeles JTP
    2. Janska A
    3. Early A
    4. Diffley JFX

    (2017) How the eukaryotic replisome achieves rapid and efficient DNA replication

    Molecular Cell 65:105–116.

    https://doi.org/10.1016/j.molcel.2016.11.017

    • PubMed
    • Google Scholar
92. 1. Zhang XY
    2. Varthi M
    3. Sykes SM
    4. Phillips C
    5. Warzecha C
    6. Zhu W
    7. Wyce A
    8. Thorne AW
    9. Berger SL
    10. McMahon SB

    (2008) The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression

    Molecular Cell 29:102–111.

    https://doi.org/10.1016/j.molcel.2007.12.015

    • PubMed
    • Google Scholar
93. 1. Zukowski A
    2. Al-Afaleq NO
    3. Duncan ED
    4. Yao T
    5. Johnson AM

    (2018) Recruitment and allosteric stimulation of a histone-deubiquitinating enzyme during heterochromatin assembly

    Journal of Biological Chemistry 293:2498–2509.

    https://doi.org/10.1074/jbc.RA117.000498

    • PubMed
    • Google Scholar

Thank you for submitting your article "FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Geeta Narlikar as Reviewing Editor and Jessica Tyler as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Vasily M Studitsky (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see the reviewers agree that your work contains new experimental findings that are important for understanding the role of H2B-Ub and histone chaperone FACT in chromatin dynamics during transcription and replication. However they also have some comments that they would like you to address experimentally to strengthen your core conclusions.

Summary:

Histone H2B ubiquitination is a highly conserved PTM coupled to active transcription. Although the enzymes that are involved in the ubiquitination and deubiquitination processes are largely known through genetics, the highly dynamic nature of this PTM has made mechanistic understanding of its function very difficult.

This manuscript reports the surprising finding that histone chaperone FACT is a key regulator of H2B deubiquitinase Ubp10 in yeast. In vitro, FACT strongly stimulates Ubp10 to deubiquitinate nucleosomes, although the underlying mechanism is not entirely clear. In vivo, ubp10-Δ and FACT mutants show synthetic effects in HU sensitivity and activation of a cryptic transcription reporter gene, suggesting that they function together in replication and transcription. Since Ubp10 was previously thought to function at subtelomere regions, these results revealed a possible new function of Ubp10 in transcriptionally active coding regions and provide a missing piece of H2Bub dynamics.

Overall the data are of high quality and presented with transparency. The results will be of interest to a broad audience in the transcription/chromatin field. However some key experimental tests are required to strengthen the conclusions as described below. Some textual clarifications are also required.

Essential revisions:

1) In Figure 2B: 2 μM Spt16/Pob3-WT and 2 μM Nhp6 was used. Is the nucleosome reorganized under these conditions? According to past studies from Formosa lab, typically reorganization requires at least 6-fold molar excess of Nhp6. More importantly does the stimulatory effect of FACT on Ubp10 action require both Spt16/Pob3 and Nhp6 in vitro? Or does FACT facilitate the deubiquitination in the absence of Nhp6 (and thus in the absence of nucleosome reorganization)? This is a critical experiment to further validate the model proposed in Figure 7.

2) The very detailed proposed model suggests that nucleosome reorganization is necessary for the deubiquitination; however, this remains to be rigorously established (see comment 1). Furthermore, nucleosome reorganization requires the presence of Nhp6 and accordingly the model explicitly implicates Nhp6 in nucleosome reorganization. However, the cycles of ubiquitination/deubiquitination are presumably coupled to ongoing transcription/replication and Nhp6 is generally not present on transcribed genes. Therefore overall, the model should be further clarified and better connected to transcription and replication.

3) Because Ubp10 has known to function in silencing, there is a formal possibility that ubp10-Δ leads to nonspecific spread of the SIR complex, which causes the observed genetic defects. Since the N-terminal regions of Ubp10 is required for its function at subtelomeres, and appears to be dispensable for FACT-dependent activity in vitro, the authors should rescue the synthetic genetic effects with Ubp10 N-terminal truncation mutants.

4) The result in Figure 4 is surprising. Previously it was reported that inactivating FACT (using a different allele) eliminates H2Bub (Fleming et al., 2008). It's possible that this difference is due to allele-specific effects. However, higher levels H2Bub have been shown to associate with better nucleosome assembly (Batta et al., 2011). Figure 6 instead suggests that nucleosome assembly is defective. This contradiction needs to be addressed with more direct evidence (see point 5 below).

5) Since westerns are notoriously difficult in discerning small differences, a more direct approach would be ChIP analysis of H2B and H2Bub levels across, for example, a long gene previously shown affected by Ubp10. This type of analysis would provide more mechanistic insights as to the effect on nucleosome integrity and relative H2Bub levels (normalized to H2B). It will also separate the effects of ubp10delta at subtelomere vs actively transcribed regions.

6) The authors propose that FACT can stimulate Ubp10 action on H2B ubiquitylated nucleosomes by either partially unfolding the nucleosome or by dissociating the H2A/H2B dimer. A straightforward way to test for either possibility would be to run the deubiquitylated products on a native gel. If the products run analogous to unmodified nucleosomes this will suggest that FACT transiently unravels the nucleosome but that the nucleosome refolds after deubiquitylation. Instead if the products run analogous to tetrasomes or hexasomes, this will suggest that Ubp10 acts on a free H2A/H2B dimer that is released by FACT action.

7) While the synthetic interaction is consistent with such a possibility, there is no direct evidence that FACT and Ubp10 function together in vivo. So we suggest that the authors make a statement in the Discussion such as: "While there is no direct evidence that FACT and Ubp10 work together in vivo, the synthetic genetic interaction and the biochemical cooperation that we observe is consistent with such a possibility".

Essential revisions:

1) In Figure 2B: 2 μM Spt16/Pob3-WT and 2 μM Nhp6 was used. Is the nucleosome reorganized under these conditions? According to past studies from Formosa lab, typically reorganization requires at least 6-fold molar excess of Nhp6. More importantly does the stimulatory effect of FACT on Ubp10 action require both Spt16/Pob3 and Nhp6 in vitro? Or does FACT facilitate the deubiquitination in the absence of Nhp6 (and thus in the absence of nucleosome reorganization)? This is a critical experiment to further validate the model proposed in Figure 7.

We thank the reviewers for suggesting this experiment. Since yeast Spt16-Pob3 does not bind stably to nucleosomes and is unable to induce nuclease sensitivity without added Nhp6, we had assumed its effects on Ubp10 activity would also have this feature. As shown in the new Figure 2—figure supplement 2 and 4, this assumption was incorrect, as Spt16-Pob3 was able to stimulate Ubp10 activity on nucleosomal H2B-Ub nearly as efficiently without Nhp6 as with it. This has implications for the model regarding how FACT affects Ubp10 activity, and we have made corresponding changes to Figure 7 as a result.

Perhaps more importantly, this result provides more insight into how FACT affects nucleosomal structure, increasing the impact of the results. Human FACT and yeast Spt16-Pob3 do not stably alter nucleosome structure, but each is able to do so with added Nhp6. However, Nhp6 is not essential for viability in yeast, whereas Spt16 and Pob3 are, and human FACT affects RNA Pol II passage under conditions where it does not produce full reorganization. It is therefore clear that Nhp6 is not required for FACT activity, but supports it in vivo and in vitro. Several labs have shown that FACT has multiple domains that interact with histone surfaces, supporting a model in which FACT binds to nucleosomes in several modes. It seems unlikely that producing the fully reorganized form detected by EMSA or restriction endonuclease digestion is the only relevant function of FACT, and we previously proposed a transient "assembly checkpoint" form of FACT:nucleosome complexes to explain some genetic and biochemical results (McCullough, et al., 2018). We now propose that one of these modes of binding along the pathway to (or returning from) full reorganization is the form that is relevant for enhancing Ubp10 activity, thus explaining the lack of a requirement for Nhp6 to produce the stably reorganized form. This is significant because it reveals a function for a partially disrupted form of nucleosomes, and because it diminishes the possibility that the stimulation of Ubp10 activity is a simple consequence of H2A-H2B dimer eviction, which does not occur in the absence of Nhp6.

2) The very detailed proposed model suggests that nucleosome reorganization is necessary for the deubiquitination; however, this remains to be rigorously established (see comment 1). Furthermore, nucleosome reorganization requires the presence of Nhp6 and accordingly the model explicitly implicates Nhp6 in nucleosome reorganization. However, the cycles of ubiquitination/deubiquitination are presumably coupled to ongoing transcription/replication and Nhp6 is generally not present on transcribed genes. Therefore overall, the model should be further clarified and better connected to transcription and replication.

The lack of an absolute requirement for Nhp6 in the stimulation of Ubp10 activity on nucleosomal substrates changes our model and our discussion of it as discussed above under point 1. We have also modified the model figure accordingly.

3) Because Ubp10 has known to function in silencing, there is a formal possibility that ubp10-Δ leads to nonspecific spread of the SIR complex, which causes the observed genetic defects. Since the N-terminal regions of Ubp10 is required for its function at subtelomeres, and appears to be dispensable for FACT-dependent activity in vitro, the authors should rescue the synthetic genetic effects with Ubp10 N-terminal truncation mutants.

Richard Gardner's lab identified Ubp10 residues 109-133 as the region necessary for interaction with the SIR complex and showed that the ubp10-∆(109-133) allele exhibited a silencing defect comparable to ubp10∆ (Reed et al., 2015). We deleted these residues from the native UBP10 locus using a markerless conversion method (Storici et al., Nat Biotechnol, 2001 19:773), combined the ubp10-∆(109-133) allele with FACT mutations, and repeated our genetic analysis. The mutation lacking the SIR-interaction domain did not affect the properties of FACT mutants in the manner observed for the full deletion of UBP10, ruling out the possibility that the phenotypes we observe are caused by the function of Ubp10 in silencing. We show the results for the pob3-L78R allele in Figure 6—figure supplement 1.

4) The result in Figure 4 is surprising. Previously it was reported that inactivating FACT (using a different allele) eliminates H2Bub (Fleming et al., 2008). It's possible that this difference is due to allele-specific effects. However, higher levels H2Bub have been shown to associate with better nucleosome assembly (Batta et al., 2011). Figure 6 instead suggests that nucleosome assembly is defective. This contradiction needs to be addressed with more direct evidence (see point 5 below).

We have found that strains with the spt16-G132D allele used by Fleming et al. have normal levels of Pob3 but reduced Spt16 under permissive conditions, whereas pob3-L78R strains have about a 10-fold reduction in Pob3 but nearly normal levels of Spt16 (VanDemark, et al., 2008). Both alleles therefore examine cells with reduced FACT levels; however, one goes from normal to very low FACT (spt16-G132D = spt16-197; acute withdrawal) while the other has chronically low FACT throughout the experiment. Further, the balance of Spt16 to Pob3 is expected to be different in the two strains. Since complete withdrawal of FACT is ultimately lethal, we think the use of chronic reduction under viable conditions is preferable to complete inactivation of Spt16 at a non-permissive temperature. Overall, we do not know why Fleming et al. observed decreased FLAG-H2B-ub levels under conditions of lethal withdrawal of FACT using the spt16-G132D allele, but we are confident that H2B-Ub increases under chronic Pob3 depletion with pob3-L78R. Moreover, we claim that pob3L78R affects Ubp10 activity in vivo, not that all FACT defects do. We have added these ideas to the manuscript (subsection “A FACT mutant strain has elevated levels of H2B-Ub”, last paragraph).

The data from Batta et al., 2011, show a more complex picture than a simple association of higher H2B-Ub with greater nucleosome occupancy. While that study shows a global decrease in nucleosome occupancy for an H2B-K123A mutation, that mutant cannot undergo cycles of H2B ubiquitination/deubiquitination and is presumably defective in FACT recruitment, all of which are thought to be important for maintaining chromatin organization (Batta et al., 2011; Fleming et al., 2008; Henry et al., 2003; Pavri et al., 2006). The decrease in global nucleosome occupancy observed in deletions of the H2B E2 ubiquitin conjugating enzyme (Rad6) and associated factor (Lge1, which binds to the E3 ligase, Bre1), and increase in global nucleosome occupancy observed in a ubp8D deletion is consistent with a role for H2B-Ub in stabilizing nucleosomes (the effects of ubp10D were not examined). However, the simple model that high global levels of H2BUb correlate with higher nucleosome occupancy is contradicted by their observation that highly transcribed genes have normal levels of nucleosome occupancy in a ubp8∆ mutant, even though these genes have markedly lower nucleosome occupancy in rad6∆ and H2B-K123A mutants (Batta et al., 2011). The relationship between global H2B-Ub levels and nucleosome occupancy is thus clearly complex. Our model that both ubiquitination and deubiquitination are important for maintaining chromatin structure and that Ubp10 and FACT function together in this process is consistent the general consensus that cycles of H2B ubiquitination and deubiquitination are critical to maintaining chromatin organization. We have added a paragraph to the Discussion (third paragraph) that summarizes these points.

5) Since westerns are notoriously difficult in discerning small differences, a more direct approach would be ChIP analysis of H2B and H2Bub levels across, for example, a long gene previously shown affected by Ubp10. This type of analysis would provide more mechanistic insights as to the effect on nucleosome integrity and relative H2Bub levels (normalized to H2B). It will also separate the effects of ubp10delta at subtelomere vs actively transcribed regions.

We believe that the western blot data we are presenting meet a much higher standard of stringency than those presented in the Fleming paper (see response to point 4 above). We agree that quantitation of western blots with histones can be difficult, but we note that we used stringent methods including a dilution series on every blot to establish linearity of all responses (with an example shown in our figure), and we report quantitative results from at least 13 independent replicates. This is in contrast to the result reported by Fleming et al. who based their conclusion on visual inspection of a single lane from a single western without quantitation or normalization. Further, their strain carried a deletion of the two H2B genes covered with a plasmid expressing FLAG-tagged H2B (H2B-FLAG). It also had a deletion of SPT16 covered with an spt16-G132D plasmid; our published work shows that each of these manipulations can lead to misregulation of at least some genes, so we instead used modifications of native loci in our experiments (unperturbed H2B genes with H2B and H2B-Ub detected by separate specific antibodies, and an otherwise unperturbed POB3 locus with just the L78R mutation.) We are therefore much more confident that this allele of FACT leads to elevated H2B-Ub than we are confident that spt16-G132D grown at the non-permissive temperature leads to its loss.

We think that western blots provide the appropriate global information we need to ask a question about the effects of Ubp10, which we propose acts genome-wide for producing global chromatin architecture, whereas Ubp8 acts locally in the context of transcription. Western blots measure the global level of H2B-Ub whereas ChIP measures local levels, which are known to vary at different sites relative to the transcription start site, as discussed in several places in this manuscript. Using ChIP to answer this question assumes that we know which loci should be affected and which should not, but this in turn assumes a model in which ubiquitination exclusively supports nucleosome stability and that loss of Ubp10 will have predictable effects localized to the same genes whose transcription is altered by this mutation. As discussed below, these assumptions may not be valid, and testing them is beyond the scope of this manuscript. Our claim is that a FACT defect can cause global loss of deubiquitination of H2B by Ubp10; we do not know or claim to know where this failure occurs.

To address the reviewers’ suggestion, we used anti-H2B-Ub ChIP to test several genes (PMA1, the HO promoter, 5' end, and 3' end, and PHO89) of different sizes whose transcript levels change in different ways in a ubp10∆ strain, but we did not see a uniform pattern of effects. H2B-Ub levels were unaffected by any mutation in PMA1, they were elevated in the promoter and 5' end of the HO gene in a pob3-L78R strain but not in a ubp10∆ strain, and they increased at the 3' end of HO in both mutants. Selected regions therefore support the model that FACT collaborates with Ubp10, but the story is clearly more complex. The initial rationale for attempting this approach was that it is difficult to use western blots to detect small changes in H2B-Ub, but ChIP introduces variation from both the antibody capture of material and using exponential PCR to detect small changes. We used 4 biological replicates of each strain and 4 technical replicates of each IP in our tests and observed more variation among the results than we did with western blots, not less. We therefore conclude that our western blot data meet the highest standard of stringency for this method, answer the question we are asking, and are more reliable for this purpose than ChIP.

The ChIP-qPCR approach is also less suitable for our needs than westerns because there is no suitable commercially available anti-H2B antibody available for ChIP, so we cannot normalize the H2B-Ub levels to the H2B levels without resorting to using an epitope tag that disrupts the phenomenon we are measuring. Our tests show that supplying FLAG-tagged H2B affects the phenotypes of FACT mutants, making this an unacceptable alternative. Overall, the only way to pursue the interesting effects we have observed is to do a ChIP-seq experiment and compare the outcomes to nucleosome occupancy and positioning by MNase-seq. We are considering initiating these studies, but they are clearly beyond the scope of this revision.

6) The authors propose that FACT can stimulate Ubp10 action on H2B ubiquitylated nucleosomes by either partially unfolding the nucleosome or by dissociating the H2A/H2B dimer. A straightforward way to test for either possibility would be to run the deubiquitylated products on a native gel. If the products run analogous to unmodified nucleosomes this will suggest that FACT transiently unravels the nucleosome but that the nucleosome refolds after deubiquitylation. Instead if the products run analogous to tetrasomes or hexasomes, this will suggest that Ubp10 acts on a free H2A/H2B dimer that is released by FACT action.

We have performed the suggested experiment, which is shown in Figure 2—figure supplement 4. The results show that the presence of FACT during the deubiquitination assay has no effect on the mobility of the three nucleosome species (with 2, 1 or 0 ubiquitins), indicating that the nucleosomes remain intact. This result suggests that FACT's effects on the nucleosomes that stimulate the activity of Ubp10 are transient and reversible.

7) While the synthetic interaction is consistent with such a possibility, there is no direct evidence that FACT and Ubp10 function together in vivo. So we suggest that the authors make a statement in the Discussion such as: "While there is no direct evidence that FACT and Ubp10 work together in vivo, the synthetic genetic interaction and the biochemical cooperation that we observe is consistent with such a possibility".

This statement has been added to the Discussion.

Author details

1. Melesse Nune

   Program in Molecular Biophysics, Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3385-3320

2. Michael T Morgan

   Program in Molecular Biophysics, Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

3. Zaily Connell

   Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Laura McCullough

   Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   Supervision, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Muhammad Jbara

   Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Hao Sun

   Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Investigation, Involved in the synthesis of mercaptolysine amino acid, a critical reagent for semisynthesis of H2Bub

   Competing interests

   No competing interests declared

7. Ashraf Brik

   Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Conceptualization, Supervision, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

8. Tim Formosa

   Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   tim@biochem.utah.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-8477-2483

9. Cynthia Wolberger

   Program in Molecular Biophysics, Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   cwolberg@jhmi.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-8578-2969

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