Solid-phase synthesis of highly repetitive chromatin assembly templates.

DNA templates for assembling chromatin model systems typically consist of numerous repeats of nucleosome positioning sequences, making their synthesis challenging. Here we describe a solid-phase strategy for generating such templates using sequential enzymatic ligation of DNA monomers. Using single nucleosome site monomers, we can either generate a twelve-nucleosome site target, or systematically access intermediate-sized templates. Using twelve nucleosome positioning site monomers, longer templates can be generated. Our synthesized templates assemble into well-defined chromatin model systems, demonstrating the utility of our solid-phase approach. Moreover, our strategy should be more widely applicable to generating other DNAs containing highly repetitive DNA sequences.

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.

Human heterochromatin protein 1α promotes nucleosome associations that drive chromatin condensation.

HP1(Hsα)-containing heterochromatin is located near centric regions of chromosomes and regulates DNA-mediated processes such as DNA repair and transcription. The higher-order structure of heterochromatin contributes to this regulation, yet the structure of heterochromatin is not well understood. We took a multidisciplinary approach to determine how HP1(Hsα)-nucleosome interactions contribute to the structure of heterochromatin. We show that HP1(Hsα) preferentially binds histone H3K9Me3-containing nucleosomal arrays in favor of non-methylated nucleosomal arrays and that nonspecific DNA interactions and pre-existing chromatin compaction promote binding. The chromo and chromo shadow domains of HP1(Hsα) play an essential role in HP1(Hsα)-nucleosome interactions, whereas the hinge region appears to have a less significant role. Electron microscopy of HP1(Hsα)-associated nucleosomal arrays showed that HP1(Hsα) caused nucleosome associations within an array, facilitating chromatin condensation. Differential sedimentation of HP1(Hsα)-associated nucleosomal arrays showed that HP1(Hsα) promotes interactions between arrays. These strand-to-strand interactions are supported by in vivo studies where tethering the Drosophila homologue HP1a to specific sites promotes interactions with distant chromosomal sites. Our findings demonstrate that HP1(Hsα)-nucleosome interactions cause chromatin condensation, a process that regulates many chromosome events.

Nucleosome interactions and stability in an ordered nucleosome array model system.

Although it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions as well as the dynamics of nucleosome stability are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail-independent, suggesting that well ordered stretches of four or fewer nucleosomes do not fully compact to the 30-nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the inter-array self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for inter-array interactions and suggesting that in vivo multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.

Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila.

Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila.

We have used immunocytochemistry to demonstrate that the EAST protein in Drosophila, which forms an expandable nuclear endoskeleton at interphase, redistributes during mitosis to colocalize with the spindle matrix proteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, we show by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which has a large NH2-terminal coiled-coil domain with the capacity for self assembly. On the basis of these findings, we propose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis.

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila.

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor.

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

Assembly interdependence among the S. cerevisiae bud neck ring proteins Elm1p, Hsl1p and Cdc12p.

In Saccharomyces cerevisiae, a complex comprising more than 20 different polypeptides assembles in a ring at the neck between the mother cell and the bud. This complex functions to coordinate cell morphology with cell division. Relatively little is known about this control system, including the physical relationships between the components of the neck ring. This study addressed the assembly interactions of three components of the ring, specifically the protein kinases Elm1p and Hsl1p and the septin Cdc12p. Specific amino acid substitutions in each of these three proteins were identified that either cause or suppress a characteristic phenotype of abnormally elongated cells and delay in the G(2)-M transition. Each protein was fused to green fluorescent protein, and its ability to localize at the neck was monitored in vivo in cells of various genotypes. Localization of Hsl1p to the neck requires Elm1p function. Elm1p localized normally in the absence of Hsl1p, although a specific point mutation in Hsl1p clearly affected Elm1p localization. The cdc12-122 mutation prevented assembly of Elm1p or Hsl1p into the neck ring. Normal assembly of Cdc12p at the neck was dependent upon Elm1p and also, to a smaller extent, on Hsl1p. Ectopic localization of Cdc12p at the bud tip was observed frequently in elm1 mutants and also, to a lesser extent, in hsl1 mutants. Thus, Elm1p is a key factor in the assembly and/or maintenance of Hsl1p, as well as at least one septin, into the bud neck ring.