Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.Eukaryotic genomic DNA is packaged into chromatin through association with positively charged histones to form the nucleosome, the structural unit of chromatin (1–3). The nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3, and H4, around which ∼146 bp of DNA winds in ∼1.65 left-handed superhelical turns (4). At this level of the DNA packaging, chromatin resembles a beads-on-a-string structure, with the nucleosome core as the beads and the linker DNA between them as the strings (5). At the next level of DNA packaging, H1 histones bind to the linker DNA and the nucleosome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cellular functions such as mitotic chromosome architecture and segregation (8), muscle differentiation (9), and regulation of gene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-amino acid unstructured C-terminal domain that is highly enriched in Lys residues. H1 stabilizes the nucleosome and facilitates folding of nucleosome arrays into higher-order structures (12–15). gH1 alone confers the same protection from micrococcal nuclease digestion to the nucleosome as the full-length H1 does (16). The N-terminal region of H1 is not important for nucleosome binding (16, 17), whereas the C terminus is required for H1 binding to chromatin in vivo (18, 19) and for the formation of a stem structure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding yeast Hho1 (23), which are both H1 homologs, have been determined at atomic resolution and show similar structures. In addition, numerous studies have indicated that gH1/gH5 binds around the dyad region of the nucleosome (14, 24), leading to many conflicting structural models for how the globular domain of H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetric and asymmetric, on the basis of the location of gH1/gH5 in the nucleosome. In the symmetric class, gH1/gH5 binds to the nucleosomal DNA at the dyad and interacts with both linker DNAs (16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to the nucleosomal DNA in the vicinity of the dyad axis and to 10 bp (27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, or is located inside the DNA gyres, where it interacts with histone H2A (35). In addition, Zhou and colleagues also characterized the orientation of gH5 in the gH5-nucleosome complex (29). The use of nonuniquely positioned nucleosomes and indirect methods may have contributed to the differences in these models (SI Appendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in particular methyl-based NMR, provides a direct approach to the structural characterization of macromolecular complexes (36, 37). We have previously assigned chemical shifts of the methyl groups of the side chains of residues Ile, Leu, and Val in the core histones (38) and the backbone amides in the disordered histone tails (39), which provide the fingerprints for investigating the interactions between H1 and the nucleosome. Here, we used NMR, along with several other methods, to determine the location and orientation of the globular domain of a stable mutant of Drosophila H1 on a well-positioned nucleosome.     

Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes

The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.The physical packaging of megabase pairs of genomic DNA stored as the chromatin fiber in eukaryotic cell nuclei has been one of the great challenges in biology (1). The limited resolution and disparate levels that can be studied by both experimental and modeling studies of chromatin, which exhibits multiple spatial and temporal scales par excellence, make it challenging to present an integrated structural view, from nucleosomes to chromosomes (2). Because all fundamental template-directed processes of DNA depend on chromatin architecture, advances in our understanding of chromatin higher-order organization are needed to help interpret numerous regulatory events from DNA damage repair to epigenetic control.At the primary structural level, the DNA makes ∼1.7 left-superhelical turns around eight core histones to form a nucleosome core. The nucleosome cores are connected by linker DNA to form nucleosome arrays. An X-ray crystal structure of the nucleosome core has been solved at atomic resolution (3), and a short, four-nucleosome array has also been solved (4). Next, at the secondary structural level, the nucleosome arrays, aided by linker histones (H1 or H5), form a compact chromatin fiber with a diameter of ∼30 nm and longitudinal compaction of 5–7 nucleosomes per 11 nm (5–8). However, evidence for 30-nm fibers in interphase nuclei of living cells has been controversial (reviewed in refs. 9 and 10). For example, whereas a distinct 30-nm fiber architecture is observed in terminally differentiated cells (11, 12), neither continuous nor periodic 30-nm fibers are observed in the nuclei of proliferating cells (13–15). However, zigzag features of the chromatin fibers are well supported by nucleosome interaction mapping in vitro (16) and in vivo (15).For chromatin architecture within metaphase chromosomes, fluorescence studies of mitotic chromosome condensation in vivo (17), cryo-EM observations of unfixed and unstained chromosomes in situ (18), and small-angle X-ray scattering (19) show no structures resembling folded 30-nm fibers and instead suggest random folding of soft polymers. Evidence is also accumulating that during chromosome condensation in mitosis, chromatin higher-order structure is dramatically altered at the global level (20) by significant increase in looping (21). A random type of looping, however, cannot explain sharp chromosomal boundaries separating the translocated genomic regions in metaphase chromosomes (22) as well as formation of highly localized fibers of transgenic DNA, up to 250 nm in diameter, detected by fluorescence imaging in vivo (17). In contrast, a hierarchical or layered looping could explain the above aspects of chromosome organization; in addition, it could help reconcile the experiments in living cells with in vitro data and determine which aspects of the secondary structure are retained in the metaphase chromosome and how these features correlate with the polymer melt model (18, 23).Here we apply the EM-assisted nucleosome interaction capture (EMANIC) technique, which captures nearest-neighbor interactions in combination with mesoscale modeling of chromatin fibers (16) to deduce chromatin architecture in interphase nuclei and metaphase chromosomes. Our results reveal persistence of the zigzag geometry as a dominant architectural motif in these types of chromatin. For metaphase chromosomes, we report a dramatic increase in longer-range interactions, consistent with intrafiber looping, quite different from that seen in compact chromatin fibers in vitro and interphase chromatin in vivo. Modeling also shows hierarchical looping for long fibers, with the loop occurrence strongly modulated by the density of linker histones. Such looping of loosely folded zigzag arrays appears to be an efficient mechanism for both condensing and unraveling the genomic material. Our hierarchical looping mechanism can also explain how distant regulatory DNA sites can be brought together naturally for genic interactions and how linker histone levels and epigenetic histone modifications can further modulate global and local chromatin architecture.     

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (“reverse topology”), but they can also be directed toward the cytosol (“normal topology”). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA⁺ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B–IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B–IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B–IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

The endosomal sorting complexes required for transport (ESCRT) proteins are an ancient and conserved membrane remodeling machinery, present in two of the three domains of life, the Archaea and Eukaryota. In humans, the ESCRTs are involved in myriad cell biological processes (1, 2) ranging from multivesicular body biogenesis (3), cytokinetic abscission (4), membrane repair (5–8), and exosome (9) and HIV-1 release (10, 11). The underlying commonality of most of these processes is that they are topologically equivalent, with scission occurring on the cytosolic and inner surface of a narrow membrane neck (“reverse topology”). The ESCRTs consist of ALIX, ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and VPS4 (12, 13). The ESCRT-III proteins (14) are most directly involved in catalyzing membrane scission (15, 16). These ESCRTs are first recruited to the neck, then the AAA⁺ ATPase VPS4 (17) is finally recruited to ESCRT-III–enriched sites prior to scission (18). VPS4 forms a hexamer (19, 20) that interacts with ESCRT-III through its N-terminal microtubule-interacting and trafficking (MIT) domain binding to the exposed C-terminal MIT domain interacting motif (MIM) domains found in some ESCRT-III proteins (21, 22). ESCRT-III together with VPS4 constitutes the minimal module to drive scission of vesicles that bud away from the cytosol (16). While ESCRTs are best known for reverse-topology membrane scission, a subset of ESCRTs, CHMP1B and IST1, can also coat the outer surface of membrane tubes, leading to a dramatic constriction in the tube (23, 24). This process is implicated in tubular endosomal traffic from the endoplasmic reticulum (ER) to lysosomes (25–27) and lipid droplets to peroxisomes (28) and the release of newly formed peroxisomes from the ER (29). These observations suggested that CHMP1B and IST1 could carry out normal topology scission; however, direct observation of this type of scission has not been reported.Twelve different ESCRT-III proteins are found in humans, which can combine in various compositions that nucleate and grow on membranes of various curvatures (23, 30, 31). ESCRT-III proteins are monomeric and are highly basic and share similar secondary core structures containing five helices (32). These proteins are in an autoinhibited closed conformation in solution (33, 34). Activation can be triggered upon binding to membranes or upstream activators, or artificially through truncation of their C-terminal elements (33, 34). Upon activation, ESCRT-III proteins polymerize into spirals (35–37) and helical tubes (24, 38). Incubation of CHMP1B with liposomes leads to formation of protein-coated tubules in vitro as shown using cryoelectron microscopy (cryo-EM) (23). This positively curved coat was initially unexpected in the ESCRT field, but subsequently has been observed more generally with combinations of CHMP2, 3, and 4 (39, 40).In reverse-topology scission, the ATPase activity of VPS4 is essential for the remodeling of ESCRT-III assemblies that lead to membrane constriction and scission (16). Polymerization of CHMP4 is considered a major driver of scission; however, this is held in check by capping with CHMP2 (41). VPS4 can solubilize CHMP2 subunits, allowing CHMP4 growth to progress, leading to scission (42). VPS4 binds to most of the MIM-containing CHMPs, including IST1 and CHMP1; however, CHMP2 is not known to have a role in normal topology scission, and CHMP1 and IST1 are not known to engage in capping. Therefore, it is not clear whether the VPS4-driven decapping mechanism established in reverse topology membrane scission has any role in normal topology scission.Spastin belongs to the same meiotic clade of AAA⁺ ATPases as VPS4 and is best known as a microtubule-severing enzyme (43, 44). Mutations of the spastin (SPG4 and SPAST) gene are the main causes in patients suffering from hereditary spastic paraplegia (45). The spastin linkage to CHMP1B and IST1 is involved in the scission of recycling endosomal tubules (27). Disruption of any of these interactions increases endosomal tubulation, mistrafficking of cargoes, and dysregulation of proper lysosomal functions (25–27). It has remained unclear whether spastin can substitute for the possible scission or disassembly functions of VPS4 with respect to CHMP1B- and IST1-containing membrane tubes, in addition to its canonical microtubule-severing activity.Recent cryo-EM reconstructions on synthetic liposomes showed that the polymerization of IST1 on the exterior of CHMP1B-coated tubes leads to a remodeling of the CHMP1B coat and tightly constricts the membrane but does not lead to membrane scission (24). Here, we used a powerful technique to assay ESCRT membrane scission in vitro by combining optical tweezers and fluorescence microscopy to visualize membrane nanotubes pulled from giant unilamellar vesicles (GUVs) (46, 47) and characterize the effect in addition of various ATPases on ESCRT dynamics. This assay allows for the formation of a positively curved membrane that mimics the membrane tubules where CHMP1B and IST1 bind. Using this highly sensitive and flexible system, we were able to reconstitute the scission reaction and delineate the roles of CHMP1B, IST1, VPS4, and spastin in normal topology scission.     

From the Cover: PNAS Plus: The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90–100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.Centromeres, the chromosomal domains that attach to spindle microtubules to segregate eukaryotic chromosomes in mitosis and meiosis, are DNA elements bound by special nucleosomes that contain a centromere-specific variant of histone H3 (cenH3). In most plants and animals, cenH3 nucleosomes are found on centromeric DNA that comprises megabases of tandemly repeated “satellite” sequences. Despite this apparent preference for repetitive DNA, a fully functional centromere, called a neocentromere, can occasionally form by assembling cenH3 nucleosomes on a single-copy DNA sequence that was not previously part of a centromere, indicating that centromere specification is epigenetic in plants and animals (for reviews, see refs. 1–4).The tandem arrays of highly repeated satellite sequences that compose most plant and animal centromeres can differ dramatically between closely related species (5), and even between different chromosomes (6–8), suggesting that satellite arrays undergo rapid evolution through expansions, contractions, gene conversions, and transpositions. Monomers of satellite repeats range in length from 5 bp in Drosophila to 1,419 bp in cattle although more than half of described monomers in 282 species have lengths between 100 and 200 bp, often regarded as approximately the length of nucleosomal DNA (6, 9). The cenH3 nucleosomes typically occupy only a portion of the satellite repeats, often in discontinuous blocks (7, 10–12), and the same or similar repeats often underlie flanking pericentromeric heterochromatin composed of conventional nucleosomes. Some of these repeats, for example African green monkey α-satellite DNA, have long been known to position conventional nucleosomes, resulting in arrays of regularly spaced nucleosomes, said to be translationally phased (13–15). Nucleosomes can occupy multiple alternative translational phases on the same satellite (16, 17). Translationally phased nucleosomal arrays have also been observed on satellites in cucumber and in several cereal species, where phasing varies among repeats and chromosomal regions (18, 19).Recently deep-sequencing technology has been applied to centromeres treated with micrococcal nuclease (MNase), which preferentially digests linker DNA between nucleosomes, to determine the positioning of cenH3 nucleosomes on satellite repeats. In human cultured cells, substantial translational phasing of CENP-A, the human cenH3, was reported on α-satellite (20). In maize, a similar approach mapped CENH3 (the name used for plant cenH3s) on the 156-bp maize centromeric satellite CentC and on two retrotransposon-derived centromeric sequences, CRM1 and CRM2 (21). Evidence for translational phasing of CENH3 on CentC and CRM1 was lacking, but 190-bp phasing was observed on CRM2. CentC was shown to have a strong periodicity of AA or TT dinucleotides about every 10 bp, which corresponds to one turn of the DNA double helix. This periodicity is thought to favor a particular orientation of the DNA toward the nucleosome core particle, based on DNA bendability, and is known as rotational phasing of nucleosomes (22–24).Rice has centromeres characterized by the 155-bp satellite sequence CentO, which is related to maize CentC (25, 26). Although some rice centromeres have megabases of CentO satellites, other evolutionarily new centromeres have little CentO, so CENH3 nucleosomes are found on both CentO and non-CentO sequences (12). For example, Cen8 is comprised of mostly non-CentO sequences and has a CentO array (CentO_8) that is spanned by a sequenced BAC (27). Centromeres like Cen8 are thought to represent an intermediate stage in centromere evolution between rare neocentromeres that form on unique sequences and mature centromeres populated by megabase-sized arrays of satellites (7, 12). Cen8 therefore presents an opportunity to compare the organization of CENH3 nucleosomes on CentO and non-CentO sequences. To that end, we used an antibody to rice CENH3 (27) to perform chromatin immunoprecipitation (ChIP) of CENH3 nucleosomes digested with MNase and sequenced the bound DNA (ChIP-Seq) to determine the positions of CENH3 nucleosomes on rice centromeres. We analyzed the sizes and positions of CENH3 nucleosomal DNA fragments on both CentO and non-CentO sequences to address the role of satellites in organizing centromeric chromatin and analyzed the sequence features of these fragments to look for evidence of nucleosome positioning signals.     

aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete, β-arrestin-2–mediated SphK1-S1PR1-Akt signaling axis

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a β-arr2–mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)–rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-arr2–dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal–regulated kinase 1/2 (ERK1/2) activation is also dependent on β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-arr2–mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, β-arr2–driven signaling pathways in caveolae.

Endothelial dysfunction, a hallmark of inflammation, is associated with the pathogenesis of vascular diseases and results in endothelial barrier disruption and increased sensitivity to apoptosis (1, 2). There are limited treatment options for improving endothelial dysfunction, which is prevalent in diseases such as sepsis, a condition with high morbidity and mortality (3, 4). Activated protein C (aPC) is a promising therapeutic that exhibits multiple pharmacological benefits in preclinical studies, including sepsis (5–7). In endothelial cells, protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR), is the central mediator of aPC cytoprotective responses, including endothelial barrier stabilization, anti-inflammatory, and anti-apoptotic activities (6). The signaling pathways by which aPC/PAR1 elicits different cytoprotective responses are poorly defined.aPC-dependent endothelial cytoprotection requires compartmentalization of PAR1 and the aPC coreceptor, endothelial protein C receptor (EPCR), in caveolin-1 (Cav1)–rich microdomains (8, 9). aPC activates PAR1 through the proteolytic cleavage of the receptor’s N-terminal arginine (R)-46 residue, which is distinct from the thrombin canonical cleavage site at (R)-41 (10). Several studies indicate that aPC/PAR1 requires β-arrestin-2 (β-arr2) to promote cytoprotection (11–13). We showed that aPC-activated PAR1 signals via β-arr2 and dishevelled-2 (Dvl2) scaffolds, and not heterotrimeric G proteins, to induce Rac1 activation and endothelial barrier protection (11). β-arr2 and Dvl2 are also required for aPC-mediated inhibition of cytokine-induced immune cell recruitment, an anti-inflammatory response (12). In addition, aPC/PAR1 stimulates Akt signaling and protects against endothelial cell death induced by tumor necrosis factor-alpha (TNF-α) and staurosporine (14, 15). However, the role of β-arr2 and Dvl2 scaffolds in mediating aPC/PAR1 anti-apoptotic responses is not known.The interaction of GPCRs with coreceptors can alter the active conformation of receptors, β-arrestin recruitment, and biased signaling (16) and is relevant to aPC/PAR1-driven endothelial cytoprotective signaling. aPC-activated PAR1 cooperates with PAR3 and sphingosine-1-phosphate receptor-1 (S1PR1) to promote cytoprotection (17–19). aPC cleaves PAR3 at a noncanonical N-terminal (R)-41 site to promote endothelial barrier protection in vitro and in vivo (19). In contrast to PAR3, aPC signals indirectly to S1PR1 to enhance basal endothelial barrier stabilization and to protect against barrier disruption (17, 18). However, the mechanism by which aPC/PAR1 transactivates S1PR1 and the role of S1PR1 in other aPC-mediated cytoprotective responses, such as cell survival, is not known.In this study, we assessed whether S1PR1 and the β-arr2 and Dvl2 scaffolds function as universal mediators of aPC/PAR1 cytoprotection by examining their function in anti-apoptotic responses. Using a combined pharmacological inhibitor and small interfering (si)RNA knockdown approach in human cultured endothelial cells, we define a β-arr2-sphingosine kinase-1 (SphK1)-S1PR1-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Our studies further demonstrate that aPC-stimulated activation of SphK1 is dependent on β-arr2 and not Dvl2, whereas neither SphK1 nor S1PR1 are required for aPC-β-arr2–induced, extracellular signal–regulated kinase 1/2 (ERK1/2) signaling. This study reveals that different aPC/PAR1 cytoprotective responses are mediated by discrete β-arr2–driven signaling pathways modulated by coreceptors localized in caveolae.     

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11–positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245–dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97– vs. Golgin-245–dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin–mediated cell polarity and cell–cell junctions.

Adenosine diphosphate (ADP) ribosylation is a protein posttranslational modification (PTM) consisting of the transfer of an ADP-ribose moiety from NAD⁺ to target amino acids that is highly conserved throughout evolution (1–3). The enzymes catalyzing this reaction, named ADP-ribosyltransferases (ARTs), first diversified in bacteria into a variety of systems involved in defensive and offensive strategies in intragenomic, intergenomic, and intraorganismal conflicts, and have been acquired by eukaryotes from these conflict systems several times throughout evolution (1, 4). In eukaryotes, ADP-ribosyltransferases are often components of core regulatory and epigenetic processes (5–7). The analysis of their eukaryotic substrates is thus likely to provide information on the organization and regulation of key cellular functions.The ARTs (8) constitute a major family of ADP-ribosyltransferases whose members catalyze ADP-ribosylation by adding either single or multiple units of the NAD⁺-deriving ADP-ribose onto target proteins [respectively, mono- and poly-ADP-ribosylation, hereafter referred to as MARylation and PARylation (9)]. MARylation of mammalian proteins was first discovered decades ago to mediate the pathogenic action of bacterial toxins in host cells (10, 11). The endogenous occurrence of this PTM in mammalian cells later became evident (11–16) and, recently, with the definition of the different enzymes catalyzing the reaction, the cellular functions it regulates are emerging (17–19).So far, eukaryotic ADP-ribosylation has been mainly studied under stress conditions, as exemplified by the role of poly (ADP-ribose) polymerase 1 (PARP1)–mediated PARylation during the DNA-damage response (20), PARP5, -12, and -13 in stress-granule formation (21–23), or PARP16 in the unfolded protein response (24, 25), while its impact on physiological cellular processes remains poorly defined.Intracellular membrane transport is emerging as a function regulated by PARPs, with particular reference to Golgi-localized PARPs, namely PARP5 and -12 (26). PARP5 (also called tankyrase) is known to regulate the delivery of the glucose transporter GLUT4 from the trans-Golgi network (TGN) to glucose-storage vesicles and thus to the plasma membrane [PM (27–30)]. PARP12, originally described to be involved in defense against viral infections (31–34), is involved in the anterograde transport of the vesicular stomatitis virus G protein (VSVG) from the TGN to the PM (21, 26, 35) and is a well-known component of stress granules, where it translocates from the Golgi upon oxidative stress (21, 23).The TGN is a major sorting station where cargoes are conveyed and sorted into distinct transport carriers for trafficking to post-Golgi compartments and to the PM (36). The different trafficking routes undertaken by individual cargoes are regulated by transport machineries, including small G proteins belonging to the ADP-ribosylation factor (Arf) and Rab families, cytosolic cargo-adaptor proteins, coat proteins, and accessory proteins, all involved in cargo “packaging” into specific transport carriers to achieve correct sorting and delivery (36–38).Here, we report that PARP12 controls the basolateral transport of a subclass of basolateral cargoes, which includes VSVG and E-cadherin, through the MARylation of Golgin-97. Moreover, we find that PARP12-mediated MARylation requires the presence of protein kinase D (PKD), a master regulator of basolateral transport (39, 40), and that it is stimulated by PKD during cargo trafficking. Traffic-activated PKD phosphorylates PARP12, activating its enzymatic activity. It thus emerged that PARP12-mediated MARylation of Golgin-97 is a component of the PKD-dependent regulatory network underlying the basolateral secretion of a select subgroup of cargo proteins, including E-cadherin. Since E-cadherin is required for the formation of proper adherens junctions and epithelial polarization, we propose that the regulatory cascade described in this study may play a role in the maintenance of cellular polarity in epithelial cells and therefore in various body functions (41, 42).     

Cohesin ATPase activities regulate DNA binding and coiled-coil configuration

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.

The cohesin complex is required for sister chromatid cohesion, DNA damage response, gene expression, and spatial organization of the genome (1, 2). Psm1/SMC1 and Psm3/SMC3 form a stable heterodimer via both hinge–hinge interaction and ATPase heads engagement upon ATP binding (3–5). Cohesin owns two ATPase domains at its globular head. Each ATPase domain contains the Walker A and Walker B consensus sequences found in most ATPases (5, 6) and several other sequence motifs, such as signature motif and D loop (7). Both ATPase domains are required for efficient loading of cohesin (8). Rad21/SCC1, the kleisin subunit with its N-terminal domain, interacts with Psm3/SMC3 coiled coils (CCs) emerging from the head, and its C-terminal domain interacts with Psm1/SMC1 head domain (9–12). Psc3/SCC3 associates with the unstructured region in the middle of Rad21/SCC1 (13–15).Mis4/SCC2/NIPBL functions as the cohesin loader (16, 17). Mis4/SCC2/NIPBL forms a harp-shaped structure (18, 19). Its N-terminal domain binds to Psm3/SMC3 coiled coils close to the head domain and its C-terminal domain binds to Psm1/SMC1 coiled coils close to the head domain (11, 15). Mis4/SCC2/NIPBL also stimulates cohesin’s ATPase activity for efficient cohesin loading (20–22).All coiled coils of SMC complexes (cohesin, condensin, and SMC5-SMC6 complex) are ∼50 nm long and are essential for their functions (23–25). SMC coiled coils contain interruptions (break sites hereafter) that disrupt the characteristic seven-residue amino acid sequence repeats, known as heptad repeats (26, 27). It has been proposed that cohesin folds around the midpoints of its coiled coils to bring the head and hinge domains into proximity (20, 28–30). However, it is still unclear how such molecular architecture of cohesin works to fulfill its function. In this study, we isolated temperature-sensitive (ts) mutants with single amino acid substitutions in the signature motif or D loop of cohesin ATPase domains, which presumably impair ATPase activity of cohesin. Then, screening of suppressor mutations that rescued the lethality caused by ATPase defects identified several hot regions in cohesin SMC subunits, which are involved in DNA binding, interaction with non-SMC subunits, or coiled-coil dynamics around midpoints. Therefore, these results coupled the dynamics of the cohesin complex with ATPase activity.