A small container of several to a few hundred microm3 (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of approximately 80 nm was found. In addition to this '80 nm fiber', a thinner '40 nm fiber' and a higher order 'loop' structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a 'coral reef structure' that also possessed the 80 nm fiber units, and, finally, into a 'tightly compacted nucleoid' that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid.

Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiledcoil with the terminal globular domains and the central hinge. Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes puri®ed from ®ssion yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holocondensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled-coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed doublestranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex.

Background: LBR is an inner nuclear membrane protein that participates in heterochromatin organization. Results: LBR recognizes specific histone modifications and induces chromatin compaction and transcriptional repression. Conclusion: LBR tethers epigenetically marked chromatin to the NE to repress transcription. Significance: This finding provides an implication of how transcriptional activities are repressed beneath the NE.

Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G 2 /M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization.

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.