Salt-urea, sulfopropyl-sepharose, and covalent chromatography methods for histone isolation and fractionation.

The histones are essential basic proteins intimately involved in most DNA-templated processes. Thus, their purification and fractionation for analysis and their use for in vitro chromatin transactions are of fundamental importance for understanding their role in chromatin structure and regulation of DNA functions. Here are described three new protocols for histone isolation from undisturbed whole cells. They avoid the conventional non-denaturing cell lysis, which affects the native posttranslational modifications of histones, and the cumbersome use of reverse-phase high-performance liquid chromatography. The three methodologies are shorter than the conventionally used protocols. The salt-urea method exploits the stability of the cell nucleus in salt solutions containing 8 M urea, whereas the cell cytoplasm and the majority of nuclear components, except H3/H4, are washed away. This protocol yields highly purified H3/H4 in a few minutes without the use of chromatography steps. The acid extraction-sulfopropyl (SP)-Sepharose protocol uses acidic solution for direct extraction of histones from undisturbed cells. Following extraction, the solution is neutralized with Tris-HCl, and run through a SP column. H2A/H2B are eluted from the SP-Sepharose at 0.8 M NaCl, with H3/H4 subsequently eluted at 2 M NaCl. This procedure yields highly purified H2A/H2B and H3/H4. Finally, covalent chromatography on thiopropyl-Sepharose (TPS) allows the separation of H3 from H4, by covalently binding H3 through its unique cysteine residue to the resin; H4 is recovered in the flow-through and wash fractions, and H3 is eluted with dithiothreitol.

Pulling chromatin apart: Unstacking or Unwrapping?

BACKGROUND: Understanding the mechanical properties of chromatin is an essential step towards deciphering the physical rules of gene regulation. In the past ten years, many single molecule experiments have been carried out, and high resolution measurements of the chromatin fiber stiffness are now available. Simulations have been used in order to link those measurements with structural cues, but so far no clear agreement among different groups has been reached. RESULTS: We revisit here some of the most precise experimental results obtained with carefully reconstituted fibers. CONCLUSIONS: We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by the stiffness of the DNA molecule and the 3D structure of the chromatin fiber.

Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X.

Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

Hho1p, the linker histone of Saccharomyces cerevisiae, is important for the proper chromatin organization in vivo.

Despite the existence of certain differences between yeast and higher eukaryotic cells a considerable part of our knowledge on chromatin structure and function has been obtained by experimenting on Saccharomyces cerevisiae. One of the peculiarities of S. cerevisiae cells is the unusual and less abundant linker histone, Hho1p. Sparse is the information about Hho1p involvement in yeast higher-order chromatin organization. In an attempt to search for possible effects of Hho1p on the global organization of chromatin, we have applied Chromatin Comet Assay (ChCA) on HHO1 knock-out yeast cells. The results showed that the mutant cells exhibited highly distorted higher-order chromatin organization. Characteristically, linker histone depleted chromatin generally exhibited longer chromatin loops than the wild-type. According to the Atomic force microscopy data the wild-type chromatin appeared well organized in structures resembling quite a lot the "30-nm" fiber in contrast to HHO1 knock-out yeast.

New nuclear partners for nucleosome assembly protein 1: unexpected associations.

Histone chaperones are important players in chromatin dynamics. They are instrumental in nucleosome assembly and disassembly and in histone variant exchange reactions that occur during DNA transactions. The molecular mechanisms of their action are not well understood and may involve interactions with various protein partners in the context of the nucleus. In an attempt to further elucidate nuclear roles of histone chaperones, we performed a proteomic search for nuclear partners of a particular histone chaperone, nucleosome assembly protein 1 (Nap1). Proteins recognized as Nap1 partners by immuno-affinity capture and Far Western blots were identified by mass spectrometry. The identified partners are known to participate in a number of nuclear processes, including DNA replication, recombination, and repair as well as RNA transcription and splicing. Finding nuclear actin among the Nap1 partners may be of particular significance, in view of actin's role in transcription, transcription regulation, and RNA splicing. We are proposing a model of how actin-Nap1 interaction may be involved in transcription elongation through chromatin. In addition, awareness of the interactions between Nap1 and Hsp70, another identified partner, may help to understand nucleosome dynamics around sites of single-strand DNA break repair. These studies represent a starting point for further investigation of Nap1 associations in human cells.

Chromatin fiber dynamics under tension and torsion.

Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism.

Histone variant H2A.Z inhibits transcription in reconstituted nucleosomes.

The existence of histone nonallelic variants has been known for more than 30 years, but only recently have we acquired significant insights into their functions. Nucleosomes containing histone variants are nonrandomly distributed in genomes and may impart different biological functions to the relevant chromatin regions. We have used the model T7 RNA polymerase to transcribe reconstituted nucleosomes containing either canonical human recombinant histones or two histone variants, H2A.Z or H3.3, whose presence has been associated with active transcription. Remarkably, in contrast to canonical and H3.3-containing nucleosomes, H2A.Z-containing nucleosomes were refractive to transcription, with residual levels of transcription determined by the sequence of the underlying DNA template. To our knowledge, this is the first example of a nucleosome that is intrinsically untranscribable.

The middle region of an HP1-binding protein, HP1-BP74, associates with linker DNA at the entry/exit site of nucleosomal DNA.

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys(97)-Lys(274)), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by approximately 25 bp. The solution structure determined by NMR revealed that the globular domain (Met(153)-Thr(237)) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.

BRCA1/BARD1 E3 ubiquitin ligase can modify histones H2A and H2B in the nucleosome particle.

BRCA1, the protein product of the Breast Cancer Susceptibility Gene (BRCA1) has been implicated in multiple pathways that preserve genome stability, including cell cycle control, DNA repair, transcription, and chromatin remodeling. BRCA1, in complex with another RING-domain protein BARD1, possesses ubiquitin-ligase activity. Only a few targets for this activity have been identified in vivo. Nucleosomal histones may also be targets in vivo since they can be modified by the BRCA1/BARD1 complex in vitro. Here we demonstrate that the BRCA1/BARD1 complex can ubiquitylate both free H2A and H2B histones and histones in the context of nucleosomal particles. These results raise the possibility that BRCA1/BARD1 can directly affect nucleosomal structure, dynamics, and function through its ability to modify nucleosomal histones.

Simplified method for recombinant linker histone H1 purification.

We report a simplified alternative protocol for purification of recombinant linker histone H1 under non-denaturing conditions. This method takes advantage of the strong affinity of H1 to DNA and comprises nucleoprotein complex extraction from the lysate of bacterial cells overexpressing the protein, followed by two ion-exchange purification steps. The purity of the protein was at least 95%; the purified H1 was tested for nucleosome binding and was successfully fluorescently labeled for further studies.

Nucleosome assembly depends on the torsion in the DNA molecule: a magnetic tweezers study.

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025-0.051 (most probably in the form of twist) stall the nucleosome assembly process.

Does BLM helicase unwind nucleosomal DNA?

RecQ helicases maintain chromosome stability by resolving several highly specific DNA structures. BLM, the protein mutated in Bloom's syndrome, is a member of the RecQ helicase family, and possesses both DNA-unwinding and strand-annealing activity. In this study, we have investigated the unwinding activity of BLM on nucleosomal DNA, the natural nuclear substrate for the enzyme. We generated a DNA template including a strong nucleosome-positioning sequence flanked by forked DNA, which is reportedly one of the preferred DNA substrates for BLM. BLM did not possess detectable unwinding activity toward the forked DNA substrate. However, the truncated BLM, lacking annealing activity, unwound it partially. In the presence of the single-stranded DNA-binding protein RPA, the unwinding activity of both the full-length and the truncated BLMs was promoted. Next, the histone octamer was reconstituted onto the forked DNA to generate a forked mononucleosome. Full-length BLM did not unwind the nucleosomal DNA, but truncated BLM unwound it partially. The unwinding activity for the mononucleosome was not promoted dramatically with RPA. These results indicate that full-length BLM may require additional factors to unwind nucleosomal DNA in vivo, and that RPA is, on its own, unable to perform this auxiliary function.

H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies.

Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochemical and biophysical analysis on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation analysis methods, we compared the properties of nucleosomes containing canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-containing nucleosomes exhibit altered linker histone binding. These properties could be physiologically significant as nucleosome positions and linker histone binding partly determine factor binding accessibility.

Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.

Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function.

CTCF and its protein partners: divide and rule?

CTCF is a ubiquitous transcription factor that is involved in numerous, seemingly unrelated functions. These functions include, but are not limited to, positive or negative regulation of transcription, enhancer-blocking activities at developmentally regulated gene clusters and at imprinted loci, and X-chromosome inactivation. Here, we review recent data acquired with state-of-the-art technologies that illuminate possible mechanisms behind the diversity of CTCF functions. CTCF interacts with numerous protein partners, including cohesin, nucleophosmin, PARP1, Yy1 and RNA polymerase II. We propose that CTCF interacts with one or two different partners according to the biological context, applying the Roman principle of governance, 'divide and rule' (divide et impera).

Parp1 localizes within the Dnmt1 promoter and protects its unmethylated state by its enzymatic activity.

BACKGROUND: Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. METHODOLOGY/PRINCIPAL FINDINGS: Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. CONCLUSIONS/SIGNIFICANCE: In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression.

The nucleosome family: dynamic and growing.

Ever since the discovery of the nucleosome in 1974, scientists have stumbled upon discrete particles in which DNA is wrapped around histone complexes of different stoichiometries: octasomes, hexasomes, tetrasomes, "split" half-nucleosomes, and, recently, bona fide hemisomes. Do all these particles exist in vivo? Under what conditions? What is their physiological significance in the complex DNA transactions in the eukaryotic nucleus? What are their dynamics? This review summarizes research spanning more than three decades and provides a new meaning to the term "nucleosome." The nucleosome can no longer be viewed as a single static entity: rather, it is a family of particles differing in their structural and dynamic properties, leading to different functionalities.

CCCTC-binding factor meets poly(ADP-ribose) polymerase-1.

CCCTC-binding factor (CTCF) is a ubiquitous Zn-finger-containing protein with numerous recognized functions, including, but not limited to, gene activation and repression, enhancer-blocking, X-chromosome inactivation, and gene imprinting. It is believed that the protein performs such a variety of functions by interacting with an array of very diverse proteins. In addition, CTCF undergoes several post-translational modifications, including poly(ADP-ribosyl)ation. The PARylated form of CTCF has recently been implicated in two important functions: gene imprinting and control of ribosomal gene transcription. Here, we summarize and critically discuss the available data on the interplay between CTCF and poly(ADP-ribosyl)ation in these two processes. We consider the newly described phenomena in the broader context of PARP's activities, including the crucial role of protein PARylation in the regulation of the genome methylation pattern.

How are nucleosomes disrupted during transcription elongation?

Chromatin structure is a powerful tool to regulate eukaryotic transcription. Moreover, nucleosomes are constantly remodeled, disassembled, and reassembled in the body of transcribed genes. Here we propose a general model that explains, in quantitative terms, how transcription elongation affects nucleosome structure at a distance as a result of the positive torque the polymerases create as they translocate along DNA templates.

Nucleosome dynamics as studied by single-pair fluorescence resonance energy transfer: a reevaluation.

Accessibility of nucleosomal DNA to protein factor binding is ensured by at least three mechanisms: post-synthetic modifications to the histones, chromatin remodeling, and spontaneous unwrapping of the DNA from the histone core. We have previously used single-pair fluorescence resonance energy transfer (spFRET) experiments to investigate long-range conformational fluctuations in nucleosomal DNA (Tomschik M, Zheng H, van Holde K, Zlatanova J, Leuba SH in Proc Natl Acad Sci USA 102(9):3278-3283, 2005). Recent work has drawn attention to a major artifact in such studies due to photoblinking of the acceptor fluorophore. We have now used formaldehyde-crosslinked nucleosomes and imaging in the presence of Trolox, an efficient triplet-state quencher that suppresses photoblinking, to reevaluate our previous conclusions. Careful analysis of the data indicates that most of the events previously characterized as nucleosome 'opening' must have corresponded to photoblinking. There is, nevertheless, evidence for the existence of infrequent, rapid opening events.