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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

The linker-protein network: control of nucleosomal DNA accessibility.

Numerous studies have recently addressed the accessibility of nucleosomal DNA to protein factors. Two popular concepts - the histone code and chromatin remodeling - consider the nucleosome as a passive entity that 'waits' to be marked by histone modifications and is 'mobilized' by ATP-dependent remodelers. Here, we propose a holistic view of the nucleosome as an active, dynamic entity, the accessibility of which is controlled by binding of different linker proteins to the DNA entry/exit site.

H2A.Z: view from the top.

For a couple of decades the chromatin field has endured undeserved neglect. Indeed, what could be so exciting about a monotonous repeating structure whose purpose in life was to package DNA? Chromatin glamour is triumphantly back, due to the realization that chromatin is a major player in the regulation of gene expression and other nuclear processes that occur on the DNA template. The dynamics of the structure that regulates transcription is itself regulated by a variety of complex processes, including histone postsynthetic modifications, chromatin remodeling, and the use of nonallelic histone variants.

Histone H1 interacts preferentially with DNA fragments containing a cisplatin-induced 1,2-intrastrand cross-link.

Cisplatin [cis-diamminedichloroplatinum(II) or cis-DDP], but not its stereoisomer transplatin, is suggested to be among the most powerful anticancer agents. It is believed that its therapeutic activity results from its interaction with DNA forming intra- and interstrand crosslinks. During our earlier investigations, we have observed a prominent preference of the linker histone H1 for binding to cis-platinated DNA (containing several different cross-links along the DNA fragment) compared with unmodified or transplatin-modified DNA.

cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines.

The phosphorylation of histone H3 is known to play a role in regulation of transcription as well as preparation of chromosomes for mitosis. Various signaling cascades induce H3 phosphorylation, particularly at genes activated by these pathways. In this study, we show that signaling can also have the opposite effect.

Chromatin fiber structure: Where is the problem now?

The structure of the "30 nm chromatin fiber", as observed in vitro, has been a matter of controversy for 30 years. Recent studies with new and more powerful techniques give some promise for resolution. However, this will not necessarily inform us as to the in vivo structure, which may be both heteromorphic and dynamic.