Histone lysine residues can be mono-, di-, or trimethylated. These posttranslational modifications regulate the affinity of effector proteins and may also impact chromatin structure independent of their role as adaptors. In order to study histone lysine methylation, particularly in the context of chromatin, we have developed a chemical approach to install analogs of methyl lysine into recombinant proteins. This approach allows for the rapid generation of large quantities of histones in which the site and degree of methylation can be specified. We demonstrate that these methyl-lysine analogs (MLAs) are functionally similar to their natural counterparts. These methylated histones were used to examine the influence of specific lysine methylation on the binding of effecter proteins and the rates of nucleosome remodeling. This simple method of introducing site-specific and degree-specific methylation into recombinant histones provides a powerful tool to investigate the biochemical mechanisms by which lysine methylation influences chromatin structure and function.

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. We show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.Chromatin-remodeling motors play essential roles in organizing the chromatin state for regulating eukaryotic genomes, yet how they carry out their myriad activities is poorly understood. Their substrate, the nucleosome, contains 147 bp of DNA wrapped in ~1.5 turns Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms around an octamer of histone proteins. Even the smallest movement of the histone octamer relative to the DNA presumably requires a coordinated process of breaking and reforming the many histone-DNA contacts. The ACF chromatin-remodeling complex exemplifies the task, as it is able to move nucleosomes to create evenly spaced nucleosomal arrays that contain equal DNA on either side of each nucleosome1-10. These evenly spaced arrays are important for packaging the underlying DNA into silent chromatin structures in vivo1-10. HHS Public AccessACF is part of the ISWI family of remodeling complexes. The ATPase subunits of ISWI complexes can move nucleosomes by themselves while the accessory subunits modulate this basic activity11-15. The human ACF complex consists of one ATPase subunit, SNF2h and one accessory subunit, Acf16,7. SNF2h is part of the SF2 family of DExx box proteins that includes helicases and nucleic acid translocases16. The ATPase domain of SNF2h has two RecA-like domains, which are thought to form a cleft within which ATP binds. SNF2h also has an alpha-helical extension comprised of three additional domains, HAND, SANT and SLIDE which are thought to play a role in bi...

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringae pathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1, pad4, eds5, and sid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance to T. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv. maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 or avrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with Psm ES4326(avrRpt2) or Psm ES4326(avrB), nahG transgenic and npr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahG and npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.Plants are frequently subjected to simultaneous insect herbivory and pathogen infection. They respond to these two different types of attackers with the induction of distinctive and overlapping subsets of secondary compounds or other defense responses involving antimicrobial or insecticidal activity. Although each type of interaction has been separately studied, the host response to the combined attack by insects and pathogens has received much less attention despite the abundance of reports indicating that pathogens and insects affect each other's performance on the host (Maleck and Dietrich, 1999;Paul et al., 2000). This study describes a model to study the three-way interactions between plants, insect herbivores, and microbial pathogens.Plants respond to insect herbivory with a complicated arsenal of defensive responses, including the synthesis of insecticidal secondary metabolites, antifeeding proteins, and/o...

Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation.ost of our understanding of bacterial physiology comes from laboratory studies of bacteria growing under nutrientrich conditions. However, in many environments, bacteria face dramatic fluctuations in nutrient conditions, including long periods of scarcity when they survive in a nonproliferative stationary state. Although eukaryotic cells and some bacteria have discrete cell cycle checkpoints, many fast-growing bacterial species have uncoupled DNA replication and cell division. They use multifork DNA replication to achieve a doubling time that is faster than the time required to copy the chromosome, giving them a competitive edge in nutrient-replete conditions. This strategy requires distinct regulatory mechanisms and comes at a considerable cost when there is a rapid downshift in nutrient availability: stalled open DNA replication forks are vulnerable to potentially lethal double-stranded DNA breaks (1). Therefore, the ability to reallocate resources under such conditions to prioritize completion of DNA replication is critical for survival. Prioritizing chromosome remodeling and compaction by starvation-specific nucleoid structural proteins is also important during such transitions because resources for DNA repair become limited in deep starvation (2-4). Operating an uncoupled cell cycle, where growth, DNA replication, and cell ...

ATP-dependent chromatin remodeling complexes enable rapid rearrangements in chromatin structure in response to developmental cues. The ATPase subunits of remodeling complexes share homology with the helicase motifs of DExx box helicases. Recent single-molecule experiments indicate that, like helicases, many of these complexes use ATP to translocate on DNA. Despite sharing this fundamental property, two key classes of remodeling complexes, the ISWI class and the SWI/SNF class, generate distinct remodeled products. SWI/SNF complexes generate nucleosomes with altered positions, nucleosomes with DNA loops and nucleosomes that are capable of exchanging histone dimers or octamers. In contrast, ISWI complexes generate nucleosomes with altered positions but in standard structures. Here, we draw analogies to monomeric and dimeric helicases and propose that ISWI and SWI/SNF complexes catalyze different outcomes in part because some ISWI complexes function as dimers while SWI/SNF complexes function as monomers.