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3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.Figure Legends, (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Saccharomyces cerevisiae Yta7 is a chromatin remodeler harboring a histone-interacting bromodomain (BRD) and two AAA+ modules. It is not well understood how Yta7 recognizes the histone H3 tail to promote nucleosome disassembly for DNA replication or RNA transcription. By cryo-EM analysis, here we show that Yta7 assembles a three-tiered hexamer with a top BRD tier, a middle AAA1 tier, and a bottom AAA2 tier. Unexpectedly, the Yta7 BRD stabilizes a four-stranded β-helix, termed BRD-interacting motif (BIM), of the largely disordered N-terminal region. The BIM motif is unique to the baker's yeast, and we show both BRD and BIM contribute to nucleosome recognition. We found that Yta7 binds both acetylated and nonacetylated H3 peptides but with a higher affinity for the unmodified peptide. This property is consistent with the absence of key residues of canonical BRDs involved in acetylated peptide recognition and the role of Yta7 in general nucleosome remodeling. Interestingly, the BRD tier exists in a spiral and a flat-ring form on top of the Yta7 AAA+ hexamer. The spiral is likely in a nucleosome-searching mode because the bottom BRD blocks the entry to the AAA+ chamber. The flat ring may be in a nucleosome disassembly state because the entry is unblocked and the H3 peptide has entered the AAA+ chamber and is stabilized by the AAA1 pore loops 1 and 2. Indeed, we show that the BRD tier is a flat ring when bound to the nucleosome. Overall, our study sheds light on the nucleosome disassembly by Yta7., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induces chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for the formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis-dependent DNA loop-extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein-protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Arabidopsis LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), a unique homolog of HP1 in Drosophila, plays important roles in plant development, growth, and architecture. In contrast to specific binding of the HP1 chromodomain to methylated H3K9 histone tails, the chromodomain of LHP1 has been shown to bind to both methylated H3K9 and H3K27 histone tails, and LHP1 carries out its function mainly via its interaction with these two epigenetic marks. However, the molecular mechanism for the recognition of methylated histone H3K9/27 by the LHP1 chromodomain is still unknown. In this study, we characterized the binding ability of LHP1 to histone H3K9 and H3K27 peptides and found that the chromodomain of LHP1 binds to histone H3K9me2/3 and H3K27me2/3 peptides with comparable affinities, although it exhibited no binding or weak binding to unmodified or monomethylated H3K9/K27 peptides. Our crystal structures of the LHP1 chromodomain in peptide-free and peptide-bound forms coupled with mutagenesis studies reveal that the chromodomain of LHP1 bears a slightly different chromodomain architecture and recognizes methylated H3K9 and H3K27 peptides via a hydrophobic clasp, similar to the chromodomains of human Polycomb proteins, which could not be explained only based on primary structure analysis. Our binding and structural studies of the LHP1 chromodomain illuminate a conserved ligand interaction mode between chromodomains of both animals and plants, and shed light on further functional study of the LHP1 protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The methyl-lysine readers plant homeodomain finger protein 20 (PHF20) and its homolog PHF20-like protein 1 (PHF20L1) are known components of the nonspecific lethal (NSL) complex that regulates gene expression through its histone acetyltransferase activity. In the current model, both PHF homologs coexist in the same NSL complex, although this was not formally tested; nor have the functions of PHF20 and PHF20L1 regarding NSL complex integrity and transcriptional regulation been investigated. Here, we perform an in-depth biochemical and functional characterization of PHF20 and PHF20L1 in the context of the NSL complex. Using mass spectrometry, genome-wide chromatin analysis, and protein-domain mapping, we identify the existence of two distinct NSL complexes that exclusively contain either PHF20 or PHF20L1. We show that the C-terminal domains of PHF20 and PHF20L1 are essential for complex formation with NSL, and the Tudor 2 domains are required for chromatin binding. The genome-wide chromatin landscape of PHF20-PHF20L1 shows that these proteins bind mostly to the same genomic regions, at promoters of highly expressed/housekeeping genes. Yet, deletion of PHF20 and PHF20L1 does not abrogate gene expression or impact the recruitment of the NSL complex to those target gene promoters, suggesting the existence of an alternative mechanism that compensates for the transcription of genes whose sustained expression is important for critical cellular functions. This work shifts the current paradigm and lays the foundation for studies on the differential roles of PHF20 and PHF20L1 in regulating NSL complex activity in physiological and diseases states., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) (also called transcription termination factor-1 interacting protein 5), a key component of the nucleolar remodeling complex, recruits the nucleolar remodeling complex to ribosomal RNA genes, leading to their transcriptional repression. In addition to its tandem plant homeodomain-bromodomain that is involved in binding to acetylated histone H4, BAZ2A also contains a methyl-CpG-binding domain (MBD)-like Tip5/ARBP/MBD (TAM) domain that shares sequence homology with the MBD. In contrast with the methyl-CpG-binding ability of the canonical MBD, the BAZ2A TAM domain has been shown to bind to promoter-associated RNAs of ribosomal RNA genes and promoter DNAs of other genes independent of DNA methylation. Nevertheless, how the TAM domain binds to RNA/DNA mechanistically remains elusive. Here, we characterized the DNA-/RNA-binding basis of the BAZ2A TAM domain by EMSAs, isothermal titration calorimetry binding assays, mutagenesis analysis, and X-ray crystallography. Our results showed that the TAM domain of BAZ2A selectively binds to dsDNA and dsRNA and that it binds to the backbone of dsDNA in a sequence nonspecific manner, which is distinct from the base-specific binding of the canonical MBD. Thus, our results explain why the TAM domain of BAZ2A does not specifically bind to mCG or TG dsDNA like the canonical MBD and also provide insights for further biological study of BAZ2A acting as a transcription factor in the future., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sister chromatid cohesion (SCC), the pairing of sister chromatids after DNA replication until mitosis, is established by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. However, how SCC is established and maintained until mitosis remains incompletely understood, and emerging evidence suggests that replication stress may lead to premature SCC loss. Here, we report that the ssDNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. After depletion of CST subunits, we observed an increase in the complete loss of SCC. In addition, we determined that CST associates with the cohesin complex. Unexpectedly, we did not find evidence of altered cohesin loading or mitotic progression in the absence of CST; however, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Because replication stress was recently shown to induce SCC loss, we hypothesized that CST may be required to maintain or remodel SCC after DNA replication fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells after exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast ( Saccharomyces cerevisiae ) and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial. Here, we show that self-interactions of the cohesin subunits Scc1/Rad21 and Scc3 occur in a DNA replication-coupled manner in both yeast and human cells. Using cross-linking MS-based and in vivo disulfide cross-linking analyses of purified cohesin, we show that a subpopulation of cohesin may exist as dimers. Importantly, upon temperature-sensitive and auxin-induced degron-mediated Eco1 depletion, the cohesin-cohesin interactions became significantly compromised, whereas deleting either the deacetylase Hos1 or the Eco1 antagonist Wpl1/Rad61 increased cohesin dimer levels by ∼20%. These results indicate that cohesin dimerizes in the S phase and monomerizes in mitosis, processes that are controlled by Eco1, Wpl1, and Hos1 in the sister chromatid cohesion-dissolution cycle. These findings suggest that cohesin dimerization is controlled by the cohesion cycle and support the notion that a double-ring cohesin model operates in sister chromatid cohesion., (© 2020 Shi et al.)

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells., (© 2020 Morales et al.)

The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. CENP-C (centromere protein C), a conserved inner kinetochore component, has been broadly characterized as a scaffolding protein and is required for the recruitment of multiple kinetochore proteins to the centromere. At its C terminus, CENP-C harbors a conserved cupin domain that has an established role in protein dimerization. Although the crystal structure of the Saccharomyces cerevisiae Mif2 CENP-C cupin domain has been determined, centromeric organization and kinetochore composition vary greatly between S. cerevisiae (point centromere) and other eukaryotes (regional centromere). Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution requires investigation. Here, we report the crystal structures of the Schizosaccharomyces pombe and Drosophila melanogaster CENP-C cupin domains at 2.52 and 1.81 Å resolutions, respectively. Although the central jelly roll architecture is conserved among the three determined CENP-C cupin domain structures, the cupin domains from organisms with regional centromeres contain additional structural features that aid in dimerization. Moreover, we found that the S. pombe Cnp3 CENP-C jelly roll fold harbors an inner binding pocket that is used to recruit the meiosis-specific protein Moa1. In summary, our results unveil the evolutionarily conserved and unique features of the CENP-C cupin domain and uncover the mechanism by which it functions as a recruitment factor., (© 2019 Chik et al.)

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Cohesin is a ring complex composed of four core subunits and seven regulatory subunits. In an effort to comprehensively identify additional cohesin-interacting proteins, we used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in cultured human cells, and we performed MS analyses on dual-affinity purifications. In addition to reciprocally identifying all known components of cohesin, we found that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs). These included diverse components of the U4/U6.U5 tri-small nuclear ribonucleoprotein complex and several splicing factors that are commonly mutated in cancer. The interaction between cohesin and splicing factors/RBPs was RNA- and DNA-independent, occurred in chromatin, was enhanced during mitosis, and required RAD21. Furthermore, cohesin-interacting splicing factors and RBPs followed the cohesin cycle and prophase pathway of cell cycle-regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors and RBPs resulted in aberrant mitotic progression. These results provide a comprehensive view of the endogenous human cohesin interactome and identify splicing factors and RBPs as functionally significant cohesin-interacting proteins., (© 2019 Kim et al.)

Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger-type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1., (© 2019 Du et al.)

The chromosomal passenger complex (CPC) is a master regulator of mitosis. CPC consists of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B and plays key roles in regulating kinetochore-microtubule attachments and spindle assembly checkpoint signaling. However, the role of CPC in sister chromatid cohesion, mediated by the cohesin complex, remains incompletely understood. Here, we show that Aurora B kinase activity contributes to centromeric cohesion protection partly through promoting kinetochore localization of the kinase Bub1. Interestingly, disrupting the interaction of INCENP with heterochromatin protein 1 (HP1) in HeLa cells selectively weakens cohesion at mitotic centromeres without detectably reducing the kinase activity of Aurora B. Thus, through this INCENP-HP1 interaction, the CPC also protects centromeric cohesion independently of Aurora B kinase activity. Moreover, the requirement for the INCENP-HP1 interaction in centromeric cohesion protection can be bypassed by tethering HP1 to centromeres or by depleting the cohesin release factor Wapl. We provide further evidence suggesting that the INCENP-HP1 interaction protects centromeric cohesion by promoting the centromere localization of Haspin, a protein kinase that antagonizes Wapl activity at centromeres. Taken together, this study identifies Aurora B kinase activity-dependent and -independent roles for the CPC in regulating centromeric cohesion during mitosis in human cells., (© 2019 Yi et al.)

The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments. When artificially tethered to centromeres, a Sgo1 mutant defective in binding protein phosphatase 2A (PP2A) is not able to support proper centromeric cohesion and CPC accumulation, indicating that the Sgo1-PP2A interaction is essential for the integrity of mitotic centromeres. We further provide evidence indicating that Sgo1 protects centromeric cohesin to create a binding site for the histone H3-associated protein kinase Haspin, which not only inhibits the cohesin release factor Wapl and thereby strengthens centromeric cohesion but also phosphorylates histone H3 at Thr-3 to position CPC at inner centromeres. Taken together, our findings reveal a positive feedback-based mechanism that ensures proper assembly of the functional inner centromere during mitosis. They further suggest a causal link between centromeric cohesion defects and chromosomal instability in cancer cells., (© 2019 Liang et al.)

The centromere is an evolutionarily conserved eukaryotic protein machinery essential for precision segregation of the parental genome into two daughter cells during mitosis. Centromere protein A (CENP-A) organizes the functional centromere via a constitutive centromere-associated network composing the CENP-T complex. However, how CENP-T assembles onto the centromere remains elusive. Here we show that CENP-T binds directly to Holliday junction recognition protein (HJURP), an evolutionarily conserved chaperone involved in loading CENP-A. The binding interface of HJURP was mapped to the C terminus of CENP-T. Depletion of HJURP by CRISPR-elicited knockout minimized recruitment of CENP-T to the centromere, indicating the importance of HJURP in CEPN-T loading. Our immunofluorescence analyses indicate that HJURP recruits CENP-T to the centromere in S/G 2 phase during the cell division cycle. Significantly, the HJURP binding-deficient mutant CENP-T 6L failed to locate to the centromere. Importantly, CENP-T insufficiency resulted in chromosome misalignment, in particular chromosomes 15 and 18. Taken together, these data define a novel molecular mechanism underlying the assembly of CENP-T onto the centromere by a temporally regulated HJURP-CENP-T interaction., (© 2019 Ding et al.)

Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions. A likely explanation for their differential regulation involves the meiosis-specific recombination proteins Hop2 and Mnd1, which are part of a highly conserved eukaryotic protein complex that participates in HR, albeit through poorly understood mechanisms. To better understand how Hop2-Mnd1 functions during HR, here we used DNA curtains in conjunction with single-molecule imaging to measure and quantify the binding of the Hop2-Mnd1 complex from Saccharomyces cerevisiae to recombination intermediates comprising Rad51- and Dmc1-ssDNA in real time. We found that yeast Hop2-Mnd1 bound rapidly to Dmc1-ssDNA filaments with high affinity and remained bound for ∼1.3 min before dissociating. We also observed that this binding interaction was highly specific for Dmc1 and found no evidence for an association of Hop2-Mnd1 with Rad51-ssDNA or RPA-ssDNA. Our findings provide new quantitative insights into the binding dynamics of Hop2-Mnd1 with the meiotic presynaptic complex. On the basis of these findings, we propose a model in which recombinase specificities for meiotic accessory proteins enhance separation of the recombinases' functions during meiotic HR., (© 2019 Crickard et al.)

The histone variant CENP-A Cse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly., (© 2018 Malik et al.)

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising ∼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the R od/ Z wilch/ Z W10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related., (© 2018 Ciossani et al.)

Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1) plays important roles in epigenetic silencing and normal mammalian development. Recently, heterozygous mutations in SMCHD1 have been reported in two disparate disorders: facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS). FSHD2-associated mutations lead to loss of function; however, whether BAMS is associated with loss- or gain-of-function mutations in SMCHD1 is unclear. Here, we have assessed the effect of SMCHD1 missense mutations from FSHD2 and BAMS patients on ATP hydrolysis activity and protein conformation and the effect of BAMS mutations on craniofacial development in a Xenopus model. These data demonstrated that FSHD2 mutations only result in decreased ATP hydrolysis, whereas many BAMS mutations can result in elevated ATPase activity and decreased eye size in Xenopus Interestingly, a mutation reported in both an FSHD2 patient and a BAMS patient results in increased ATPase activity and a smaller Xenopus eye size. Mutations in the extended ATPase domain increased catalytic activity, suggesting critical regulatory intramolecular interactions and the possibility of targeting this region therapeutically to boost SMCHD1's activity to counter FSHD., (© 2018 Gurzau et al.)

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins ( i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type-I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaA-enzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type-I cohesin-dockerin specificity in A. cellulolyticus Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA- and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both, of the ligand-binding sites of ScaB, whereas attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The mammalian SWI/SNF chromatin remodeling complex is a heterogeneous collection of related protein complexes required for gene regulation and genome integrity. It contains a central ATPase (BRM or BRG1) and various combinations of 10-14 accessory subunits (BAFs for B RM/BRG1 A ssociated F actor s ). Two distinct complexes differing in size, BAF and the slightly larger polybromo-BAF (PBAF), share many of the same core subunits but are differentiated primarily by having either AT-rich interaction domain 1A/B (ARID1A/B in BAF) or ARID2 (in PBAF). Using density gradient centrifugation and immunoprecipitation, we have identified and characterized a third and smaller SWI/SNF subcomplex. We termed this complex GBAF because it incorporates two mutually exclusive paralogs, GLTSCR1 (glioma tumor suppressor candidate region gene 1) or GLTSCR1L (GLTSCR1-like), instead of an ARID protein. In addition to GLTSCR1 or GLTSCR1L, the GBAF complex contains BRD9 (bromodomain-containing 9) and the BAF subunits BAF155, BAF60, SS18, BAF53a, and BRG1/BRM. We observed that GBAF does not contain the core BAF subunits BAF45, BAF47, or BAF57. Even without these subunits, GBAF displayed in vitro ATPase activity and bulk chromatin affinity comparable to those of BAF. GBAF associated with BRD4, but, unlike BRD4, the GBAF component GLTSCR1 was not required for the viability of the LNCaP prostate cancer cell line. In contrast, GLTSCR1 or GLTSCR1L knockouts in the metastatic prostate cancer cell line PC3 resulted in a loss in proliferation and colony-forming ability. Taken together, our results provide evidence for a compositionally novel SWI/SNF subcomplex with cell type-specific functions., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCβ1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the Pax7 promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB-topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA. This effect does not require the catalytic activity of topoisomerase IV. Cells carrying a mukB mutant allele that encodes a protein that does not interact with topoisomerase IV exhibit severe nucleoid decompaction leading to chromosome segregation defects. These findings suggest that the MukB-topoisomerase IV complex may provide a scaffold for DNA condensation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G 2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

DNA replication greatly enhances expression of the herpes simplex virus 1 (HSV-1) γ2 late genes by still unknown mechanisms. Here, we demonstrate that 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK9, suppresses expression of γ2 late genes with an IC 50 of 5 μm, which is at least 10 times lower than the IC 50 value required for inhibition of expression of early genes. The effect of DRB could not be explained by inhibition of DNA replication per se or loading of RNA polymerase II to late promoters and subsequent reduction of transcription. Instead, DRB reduces accumulation of γ2 late mRNA in the cytoplasm. In addition, we show that siRNA-mediated knockdown of the transcription factor SPT5, but not NELF-E, also gives rise to a specific inhibition of HSV-1 late gene expression. Finally, addition of DRB reduces co-immunoprecipitation of ICP27 using an anti-SPT5 antibody. Our results suggest that efficient expression of replication-dependent γ2 late genes is, at least in part, regulated by CDK9 dependent co- and/or post-transcriptional events involving SPT5 and ICP27., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

To ensure correct spatial and temporal patterning, embryos must maintain pluripotent cell populations and control when cells undergo commitment. The newly identified nucleoprotein Akirin has been shown to modulate the innate immune response through epigenetic regulation and to play important roles in other physiological processes, but its role in neural development remains unknown. Here we show that Akirin2 is required for neural development in Xenopus and that knockdown of Akirin2 expands the expression of the neural progenitor marker Sox2 and inhibits expression of the differentiated neuronal marker N-tubulin. Akirin2 acts antagonistically to Geminin, thus regulating Sox2 expression, and maintains the neural precursor state by participating in the Brg1/Brm-associated factor (BAF) complex mediated by BAF53a. Additionally, Akirin2 also modulates N-tubulin expression by acting upstream of neuronal differentiation 1 (NeuroD) and in parallel with neurogenin-related 1 (Ngnr1) during terminal neuronal differentiation. Thus, our results reveal a novel model in which Akirin2 precisely coordinates and temporally controls Xenopus neural development., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In Drosophila and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depleted Drosophila nuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Production of hemoglobin during development is tightly regulated. For example, expression from the human β-globin gene locus, comprising β-, δ-, ϵ-, and γ-globin genes, switches from ϵ-globin to γ-globin during embryonic development and then from γ-globin to β-globin after birth. Expression of human ϵ-globin in mice has been shown to ameliorate anemia caused by β-globin mutations, including those causing β-thalassemia and sickle cell disease, raising the prospect that reactivation of ϵ-globin expression could be used in managing these conditions in humans. Although the human globin genes are known to be regulated by a variety of multiprotein complexes containing enzymes that catalyze epigenetic modifications, the exact mechanisms controlling ϵ-globin gene silencing remain elusive. Here we found that the heterochromatin protein HP1γ, a multifunctional chromatin- and DNA-binding protein with roles in transcriptional activation and elongation, represses ϵ-globin expression by interacting with a histone-modifying enzyme, lysine methyltransferase SUV4-20h2. Silencing of HP1γ expression markedly decreased repressive histone marks and the multimethylation of histone H3 lysine 9 and H4 lysine 20, leading to a significant decrease in DNA methylation at the proximal promoter of the ϵ-globin gene and greatly increased ϵ-globin expression. In addition, using chromatin immunoprecipitation, we showed that SUV4-20h2 facilitates the deposition of HP1γ on the ϵ-globin-proximal promoter. Thus, these data indicate that HP1γ is a novel epigenetic repressor of ϵ-globin gene expression and provide a potential strategy for targeted therapies for β-thalassemia and sickle cell disease., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequence-specific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcript ion factors (Cdx1, Cdx2, and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI-SNF complex and make direct contact with Brg1, a catalytic member of SWI-SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at some of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI-SNF chromatin remodeling activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The assembly of one of Nature's most elaborate multienzyme complexes, the cellulosome, results from the binding of enzyme-borne dockerins to reiterated cohesin domains located in a non-catalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting in the gastrointestinal tract of mammals., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein acetylation is a prevalent posttranslational modification that is regulated by diverse acetyltransferase enzymes. Although histone acetyltransferases (HATs) have been well characterized both structurally and mechanistically, far less is known about non-histone acetyltransferase enzymes. The human ESCO1 and ESCO2 paralogs acetylate the cohesin complex subunit SMC3 to regulate the separation of sister chromatids during mitosis and meiosis. Missense mutations within the acetyltransferase domain of these proteins correlate with diseases, including endometrial cancers and Roberts syndrome. Despite their biological importance, the mechanisms underlying acetylation by the ESCO proteins are not understood. Here, we report the X-ray crystal structure of the highly conserved zinc finger-acetyltransferase moiety of ESCO1 with accompanying structure-based mutagenesis and biochemical characterization. We find that the ESCO1 acetyltransferase core is structurally homologous to the Gcn5 HAT, but contains unique additional features including a zinc finger and an ∼40-residue loop region that appear to play roles in protein stability and SMC3 substrate binding. We identify key residues that play roles in substrate binding and catalysis, and rationalize the functional consequences of disease-associated mutations. Together, these studies reveal the molecular basis for SMC3 acetylation by ESCO1 and have broader implications for understanding the structure/function of non-histone acetyltransferases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Properly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Borealin, a member of the chromosomal passenger complex, plays a key regulatory role at centromeres and the central spindle during mitosis. Loss of Borealin leads to defective cell proliferation and early embryonic lethality. The in vivo functions of Borealin in mammalian postnatal development, tissue homeostasis, and tumorigenesis remain elusive. We specifically analyzed the role of Borealin in regulating postnatal liver development, damage-induced liver regeneration, and liver carcinogenesis using mice carrying conditional Borealin alleles. Perinatal loss of Borealin caused increased genome ploidy and enlarged cell size in hepatocytes, likely due to the impaired function of the chromosomal passenger complex in mitosis. Borealin deletion also showed attenuated expansion of Sox9 + HNF4α + progenitor-like cells in liver regeneration during 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced liver injury. Moreover, ΔN90-β-Catenin and c-Met-induced hepatocarcinogenesis development was largely impeded by Borealin deletion. These findings indicate that Borealin plays a key role in liver development, regeneration, and tumorigenesis and suggests that Borealin could be a potential target for related liver diseases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood however. Here, we show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in S phase, consistent with a role of Naa50 in cohesion establishment. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro Because NatA and Naa50 exhibit distinct substrate specificity, we propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We show how enhancers of macrophage-specific genes are rendered accessible in differentiating macrophages to allow their induction in mature cells in response to an appropriate stimulus. Using a lentiviral knockdown approach in primary differentiating macrophages from mouse bone marrow, we demonstrate that enhancers of Il12b and Il1a are kept relatively lowly occupied by nucleosomes and accessible through recruitment of the nucleosome remodeler BAF/PBAF. Our results using an inducible cell line that expresses an estrogen receptor fusion of the macrophage-specific transcription factor PU.1 (PUER) show that BAF/PBAF recruitment to these enhancers is a consequence of translocation of PUER to the nucleus in the presence of tamoxifen, and we speculate that remodeler recruitment may be directly mediated by PU.1. In the absence of BAF/PBAF recruitment, nucleosome occupancy at the enhancer of Il12b (and to a lesser extent at Il1a) reaches high levels in bone marrow-derived macrophages (BMDMs), and the enhancers are not fully cleared of nucleosomes upon LPS induction, resulting in impaired gene expression. Analysis of Il12b expression in single cells suggests that recruitment of the remodeler is necessary for high levels of transcription from the same promoter, and we propose that remodelers function by increasing nucleosome turnover to facilitate transcription factor over nucleosome binding in a process we have termed "remodeler-assisted competition.", (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cohesin, a multi-subunit protein complex involved in chromosome organization, is frequently mutated or aberrantly expressed in cancer. Multiple functions of cohesin, including cell division and gene expression, highlight its potential as a novel therapeutic target. The SMC3 subunit of cohesin is acetylated (ac) during S phase to establish cohesion between replicated chromosomes. Following anaphase, ac-SMC3 is deacetylated by HDAC8. Reversal of SMC3 acetylation is imperative for recycling cohesin so that it can be reloaded in interphase for both non-mitotic and mitotic functions. We blocked deacetylation of ac-SMC3 using an HDAC8-specific inhibitor PCI-34051 in MCF7 breast cancer cells, and examined the effects on transcription of cohesin-dependent genes that respond to estrogen. HDAC8 inhibition led to accumulation of ac-SMC3 as expected, but surprisingly, had no influence on the transcription of estrogen-responsive genes that are altered by siRNA targeting of RAD21 or SMC3. Knockdown of RAD21 altered estrogen receptor α (ER) recruitment at SOX4 and IL20, and affected transcription of these genes, while HDAC8 inhibition did not. Rather, inhibition of HDAC8 delayed cell cycle progression, suppressed proliferation and induced apoptosis in a concentration-dependent manner. We conclude that HDAC8 inhibition does not change the estrogen-specific transcriptional role of cohesin in MCF7 cells, but instead, compromises cell cycle progression and cell survival. Our results argue that candidate inhibitors of cohesin function may differ in their effects depending on the cellular genotype and should be thoroughly tested for predicted effects on cohesin's mechanistic roles., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cse4, a histone H3-like centromeric protein, plays critical functions in chromosome segregation. Cse4 level is tightly regulated, but the underlying mechanism remains poorly understood. We employed a toxicity-based screen to look for the degradation components involved in Cse4 regulation. Here, we show that the F-box containing protein Rcy1 is required for efficient Cse4 turnover as Cse4 degradation is compromised in yeast cells lacking RCY1 Excessive Cse4 accumulation in rcy1Δ cells leads to growth retardation. Furthermore, the deletion of RCY1 is tied to enhanced chromosome instability and temperature-sensitive cell growth. Our results reveal the involvement of Rcy1 in chromosome regulation and another regulatory pathway controlling the Cse4 level and activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)