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The atomic force microscope (AFM) can be used both to manipulate molecules and to make measurements on individual molecules. Here, we describe manipulation of chromatin constructs as an example of the first type of measurement, and electronic measurements on single molecules as an example of the second type.An AFM was used to both image and stretch single synthetic chromatin fibers consisting of twelve core nucleosomes with no linker histones. Peaks in the force-curves are attributed to sequential detachment of nucleosomes from the glass support (Figure 1). The short distances between peaks and reversibility of the pulling process show that the nucleosomes remain intact even at tensions on the order of 350 picoNewtons (pN). This is more than an order of magnitude larger than the force required to de-spool histone octamers from the nucleosomal DNA in laser optical tweezer measurements made with longer molecules, suggesting that loading rates and sample size are important factors in determining the force required to break inter-molecular bonds.

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.

DNA in the eukaryotic nucleus is not naked. Instead, it is complexed at frequent intervals with an equal molecular mass of histones to form nucleosomes. In the nucleosome, ∼146 bp of DNA are wrapped around a core histone octamer composed of two copies each of histones H4, H3, H2B, and H2A. The family of linker histones (H1, H5 and H1°) bind to the DNA between successive nucleosomes and help maintain the three-dimensional arrangement of nucleosomes within the chromatin fiber. AFM studies of chromatin fibers with the histones either selectively trypsinized or selectively reconstituted demonstrate a specific role for the H3 N-termini in maintaining fiber structure, in conjunction with the 80 amino acid linker histone globular domain. These AFM results structurally agree with the location of the H3 N-termini in the histone octamer or the nucleosome core particle.

Recently, a quantum leap has been achieved in the analysis of DNA and proteins through the advent of the biochip technology. This technology is a product of a broad interdisciplinary approach combining biochemical analysis, semiconductor manufacturing and computer software. Biochips can be defined as miniaturized ordered arrays of macro molecules or pieces thereof that are immobilized in a precise spatial manner on support media and can be used in highly automated, large-scale and high-throughput fashion to analyze biological material. The biochip can be used in a wide variety of areas related to basic research and can find versatile applications in almost all areas of human activities connected to biotechnology, medicine, agriculture, and environment monitoring and bioremediation.The power of the technology has already been demonstrated in areas like gene sequencing and proofreading, detection of single-nucleotide mutation and polymorphism, identification of genes, identification of viruses and microorganisms, gene expression analysis, analysis of sequencespecific ligands and proteins, and others.